CN109425670A

CN109425670A - A method of teams and groups' degree of fatigue is detected based on human urine

Info

Publication number: CN109425670A
Application number: CN201710781324.7A
Authority: CN
Inventors: 陈振玲; 刘永锁; 李丽丽; 姜薇; 徐先发; 许海山; 王伟; 王妍; 刘铁兵; 王雷; 朱安林; 杨剑
Original assignee: CIVIL AVIATION MEDICAL CENTER CIVIL AVIATION ADMINISTRATION OF CHINA
Current assignee: CIVIL AVIATION MEDICAL CENTER CIVIL AVIATION ADMINISTRATION OF CHINA
Priority date: 2017-09-01
Filing date: 2017-09-01
Publication date: 2019-03-05
Anticipated expiration: 2037-09-01
Also published as: CN109425670B

Abstract

The present invention relates to a kind of methods based on human urine detection teams and groups' degree of fatigue, and described method includes following steps: the urine specimen of step 1. acquisition crewmember；The sample that step 2. acquires step 1 pre-processes；Step 3. detects the pretreated sample of step 2 using liquid chromatography-mass spectrometry；The data that step 4. obtains step 3 detection carry out processing and threshold value is set, and filter out potential source biomolecule marker；The potential source biomolecule marker that step 4 filters out is compared step 5. with degree of fatigue marker, determines the degree of fatigue for being detected crewmember.The present invention realizes the determination that degree of fatigue detects marker by the method for small molecule metabolism group, and is able to carry out the judgement of degree of fatigue, and this method universality is good, accuracy and with a high credibility.

Description

A method of teams and groups' degree of fatigue is detected based on human urine

Technical field

The invention belongs to technical field of biological, and in particular to a kind of to detect teams and groups' degree of fatigue based on human urine Method.

Background technique

The history of human research's fatigue can trace back to the World War I before a century earliest, use ergonomics side first Method research fatigue problem utilizes the index characterizations such as labor productivity, job performance fatigue.

1970s, people start to notice that driver fatigue is easy to cause traffic accidents and accident proneness, To which fatigue be connected with safety accident and accident proneness.The target development of fatigue study is prevention due to caused by fatigue Accident and accident proneness ensure safety.With highly popular, the labor intensity reduction of industrial automation, brainfag (mental Fatigue) become the main tired form in industrial production.Corresponding physical fatigue (physical fatigue), Also referred to as muscular fatigue (muscle fatigue), which refers to, occurs to become powerless in the DOMS and muscle of part.This body Body fatigue is after today's society occurs mainly in strenuous exercise, also referred to as sports fatigue.In mobile, we are mentioned tired When labor is not indicated especially, brainfag is referred both to.

In the industrial production, fatigue is associated with safety in production, therefore different safety in production posies, for tired tolerance It has differences.For example, in the post of turnpike driving haulage vehicle, since speed is fast, to ensure transportation safety requirement High driver focuses on, has sound response power, and it is all flagrant for slight fatigue occur to driver.For another example International Civil Aviation Organization (International Civil Aviation Organization) describes fatigue are as follows: due to sleep Missing, awakening time extend, circadian rhythm changes, (mental or/and physical strength) workload, these single factor tests or multifactor lead The human body alertness of cause declines and executes the physiology shape of a kind of psychology and physiological function reduction of safety-related Duty Capacity decline State.Therefore, judgement tired in industry security production is, security sensitive relevant to personnel's safe post Duty Capacity of execution To the tolerance level of degree of fatigue, i.e. influence of the fatigue level of human body to production safety is that industry security production is closed all the time in post One of research hotspot of note.

The research of fatigue problem is also expanded in the continuous improvement paid close attention to people social safety by ergonomics research To subjects such as psychology, Neuscience, hypnosphy, Physiology and biochemistries.The method that each subject establishes corresponding detection fatigue, such as the heart Stamford Sleepiness Scale, Visual analogue scale etc. of the Neo-Confucianism based on subjective feeling, there are also the prestige based on cognitive ability detection fatigue This Kang Xing card sorting tests (WSCT), Tower of London test (TOL) etc..Subjective scale is interfered vulnerable to mood and other factors, Parameter index setting is relatively difficult in recognition tests, " ceiling " or " floor " effect often occurs, causes these scales or soft The reliability and validity of part are not satisfactory enough；For another example nerve and sleep science are established based on electroencephalogram variation detection fatigue method, It is proposed occur fatigue when delta and theta wave significantly increases, due to Brain Diagnostic Apparatus complexity, it is difficult to be applied to on-site test；Again It is such as based on occurring yawning when human-body fatigue, eye movement variation such as blink, eyelid closure physiological reaction, attempts to establish corresponding detection Method.Automobile Transportation office, United States highways management board issues a technical report in October, 1998 and points out, with brain electricity Detection, head position detection, blink detection are compared, and eyelid closure (PERCLOS) detection fatigue has more accurate and stablizes Testing result.But the detection of eyelid closure is interfered by glasses, ambient light angle and brightness, human eye angle etc., it is difficult to be pushed away Wide application.So far, people understand seldom tired biochemical mechanism, and the explaination of tired biochemical mechanism will be helpful to exploitation and be based on Objective, stable, quickly detection degree of fatigue the method for biochemical indicator.

It is a kind of method that people always try to using biochemical indicator detection degree of fatigue, such as detects some hormones, peptide, Research finds that cytidine (Cytidine) and adrenal cortex class ester alcohol (Corticoid) concentration are related with operating pressure； Also detect substances related with Shui Mian, biological rhythm such as glycopeptide (Glycopeptides)；But Borbely etc. points out these chemical combination Object has the evidence of the data deficiency consistency of correlation with fatigue.In short, although people save from operating pressure, sleep, biology The multi-angles such as rule have attempted multiple compounds metabolism and degree of fatigue correlation research, however do not find any human body generation yet Thanking compound and degree of fatigue has correlation.

Metabolism group is the new branch of science of metabolic characteristics in a system research body, mainly uses nuclear magnetic resonance, gas phase color Spectrum-mass spectrum, liquid chromatography-mass spectrography etc. test and analyze means combination Chemical Measurement, systematically explore, find and find human body by To characteristic index, that is, biomarker of its body fluid metabolic when the influences such as disease, drug, in fields such as medical diagnosis on disease, medicament research and developments It is widely applied.For example, Mapstone of Georgetown University, the U.S. etc. is using LC Mass (analysis of liquid matter) Metabolism group method, screening goes out 10 kinds of feature biomarkers, for detecting alzheimer's disease, can face in the disease 2-3 is diagnosed before bed symptom, and accuracy rate can reach 90% or more；For another example, Naviaux etc. is ground using liquid matter analysis method Chronic fatigue syndrome metabolic characteristics is studied carefully, discovery male patient there are 8 kinds of metabolins, and 13 kinds of metabolins of female patient occur significant It sexually revises, Subject characteristics' working curve (Receiver operator characteristic curve, ROC) analysis knot Fruit shows that accuracy rate of diagnosis reaches 94% (male) and 96% (women)；Professor Xu Guowang etc. analyzed using liquid matter to depression and The blood and urine of over fatigue rat model have carried out metabolism group research, the results showed that two groups of rats and control rats phase Than anomalous variation occurs in metabolism.Armstrong etc. samples two dimensional NMR energy disperse spectroscopy to chronic fatigue syndrome patients serum Metabolism group research is carried out, the results showed that case group serum glutamine and ornithine content are substantially less than control group.

In above-mentioned document report, case group is all made of for various diseases and control group is grouped comparison, but due to tired Labor is a kind of physiological change that can restore, and existing literature method is not suitable for the screening of degree of fatigue associated biomarkers. Chen etc. utilizes liquid chromatography-mass spectrometry in the literature, has screened 3 kinds of fatigues to the urine specimen of air traffic control person Associated biomarkers, but the concept of degree of fatigue is not proposed, furthermore fail to disclose how quantization detection degree of fatigue, accurately Ground goes the method for judging whether fatigue and degree of fatigue occur.

In the course of more than 100 years of fatigue study, it was recognized that there are different intensity, i.e. degree of fatigue for fatigue, and And it attempts divide degree of fatigue, characterize, detecting.Such as psychologic Shi Tanfu Sleepiness Scale, wherein by people by complete Waking state is to almost having a dream, it appears that the state demarcation that next second will fall asleep is at 7 level of fatigue, it is desirable that subject is logical It crosses subjective feeling and carries out selection division.So far, there is not yet detecting the report of degree of fatigue using objective indicator.Tired journey Degree is related to potential job safety executive capability in industrial production, such as turnpike driving person continuously drives 2-3 hours and occurs gently Degree fatigue, needs to rest and restore 30-60 minutes, could continue to be engaged in this work for needing significant attention security risk high Make；And general office work office worker then can be 4-6 hours with continuous work, even if safety wind will not still be generated by slight fatigue occur Danger.Therefore degree of fatigue detection can provide strong new technology and new tool to production safety risk management and control.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, is established using small molecule metabolism group method a kind of using liquid Method of the phase chromatography mass spectrometry based on human urine detection teams and groups' degree of fatigue.It in turn can according to the assessment result of degree of fatigue To carry out workload assessment, system of arranging an order according to class and grade assessment in production, whether pre-job check and evaluation can be on duty, detect and use in hilllock It adjusts in the tired risk of discovery and in time, rear check and evaluation post workload on duty etc..It is examined for degree of fatigue in industrial production It surveys and monitors, manage tired risk, guarantee safe production and new technology and method is provided.

On a large amount of experiment basis, proposing can be carried out the present inventor by the expression of tired associated biomarkers New thought degree of fatigue detection and divided, and quantization detection degree of fatigue is established in the present invention, accurately go judgement to be The no method for fatigue and degree of fatigue occur.

The present invention is achieved through the following technical solutions:

A method of teams and groups' degree of fatigue being detected based on human urine, which is characterized in that the method includes walking as follows It is rapid:

The urine specimen of step 1. acquisition crewmember；

The sample that step 2. acquires step 1 pre-processes；

Step 3. detects the pretreated sample of step 2 using liquid chromatography-mass spectrometry；

The data that step 4. obtains step 3 detection carry out processing and threshold value is set, and filter out potential source biomolecule marker；

The potential source biomolecule marker that step 4 filters out is compared step 5. with degree of fatigue marker, determines tested Survey the degree of fatigue of crewmember.

According to the present invention, in step 1,

The urine specimen derives from the crewmember of health, when the crewmember is women, does not preferably exist The women in menstrual period.

According to the present invention, the acquisition step includes acquiring non-tired sample and degree of fatigue sample.

According to the present invention, the acquisition step of sample includes: before crewmember goes to work work or simulated operation induces fatigue Before, its urine specimen is acquired as non-tired sample；According to degree of fatigue, after crewmember's work or simulated operation induction is tired Its urine specimen is collected after labor as degree of fatigue sample.

According to the present invention, it the stream time length, work position of each member, workload and arranges an order according to class and grade in the teams and groups Situation etc. is same or similar.

The teams and groups can be for example driver, controller, flight-line maintenance personnel or teams and groups of healthcare givers's composition etc.；

According to the present invention, the teams and groups can be the sample size group of 5 people or more, such as the sample size group of 8-20 people, such as 8- The sample size group of 10 people.

According to the present invention, the sample collection procedure of step 1 further includes after having acquired urine specimen, by sample not high Cryo-conservation is carried out under the conditions of -20 DEG C.

Such as urine specimen is sub-packed in sterile centrifugation tube and is stored in -80 DEG C.

Appropriate antibiotic can also be added into sample during cryo-conservation, avoid causing part metabolin because of bacterium Decomposition；Such as the sodium azide that mass concentration is 0.05-0.1% is added into sample.

According to the present invention, in the step 2,

Pre-treatment step includes: to be centrifuged urine specimen, take supernatant and optionally by supernatant be diluted to be suitable for after The aqueous solution of continuous step analysis detection concentration, such as sample is diluted using the water of 1-3 times of volume.

Urine specimen is pre-processed for example, by using following steps:

Urine specimen is taken out before analysis detection and is placed to room temperature, at 4 DEG C, 12000rpm is centrifuged 5 minutes, takes supernatant The dilution of 100 μ L water is added in 100 μ L.

According to the method for the present invention, in step 3,

The liquid chromatography-mass spectrometry is preferably sewage sludge method.

The detection sample is the degree of fatigue sample and non-tired sample of teams and groups；

The liquid chromatogram can use the preferred HILIC column of polarity chromatographic column, the preferred C of low pole chromatographic column₁₈Column and non-pole Property chromatographic column preferred PFPP column separation detection is carried out to polarity, low pole and the non-polar component in urine respectively.

When the liquid chromatogram use polarity chromatographic column separation detection polar compound when, mass spectrography preferably using detection just from Subpattern；When the liquid chromatogram is using low pole and non-polar column separation detection low pole and non-polar component, mass spectrography Using cation and anion both of which.

Preferably, using the liquid-phase condition of polarity HILIC chromatographic column are as follows: UPLC BEH Amide HILIC column [(2.1-4.6)mm×(100-250)mm,1.5-5μm]；

Mobile phase is that A phase composition is volume fraction 95-100% acetonitrile or methanol and volume fraction is that 0-5% contains 0.1- The aqueous solution of 0.5% formic acid or acetic acid or trifluoroacetic acid, B phase composition are to contain 0.1-0.5% formic acid or acetic acid or trifluoroacetic acid Aqueous solution；

Column temperature is 20-40 DEG C, sample volume 0.5-5.0 μ L, flow rate of mobile phase 0.3-1.0mL/min.

Preferably, using low pole C₁₈The liquid-phase condition of chromatographic column are as follows: UPLC CSH C₁₈Column [(2.1-4.6) mm × (100-250)mm,1.5-5μm)]；

Mobile phase A phase composition is the aqueous solution containing 0.1-5% formic acid or acetic acid or trifluoroacetic acid, and B phase composition is acetonitrile Or methanol；

Preferably, liquid chromatography uses the liquid-phase condition of nonpolarity PFPP chromatographic column are as follows: UPLC HSS PFPP column [(2.1-4.6)mm×(100-250)mm,1.5-5μm)]；

Mobile phase is that A phase composition is the aqueous solution containing 0.1-5% formic acid or acetic acid or trifluoroacetic acid, and B phase composition is first Alcohol or acetonitrile；

Column temperature is 20-40 DEG C, sample volume 0.3-5.0 μ L, flow rate of mobile phase 0.3-1.0mL/min.

The testing conditions of the mass spectrography are as follows:

When being detected using cation ionization mode, mass range is set as 50-1200m/z full scan mode.EFI Mist ionizer capillary voltage is 2500-3500, and preferably 3000V, cone voltage is 20-40 V, preferably 30V.Drying gas is Nitrogen, desolvation flow velocity are 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100℃.Concentration is used to calibrate internal standard as mass number for the leucineenkephalin (leucine enkephalin) of 0.2ng/mL, Calibration mass of ion is 556.2771Da ([M+H]⁺)。

When being detected using two kinds of ionization modes of cation and anion, it is complete that mass range is set as 50-1200 m/z Scan pattern；Electro-spray ionization device capillary voltage be 2000-3500, preferably 3000V (cation) or 2200V (bear from Son), cone voltage is 20-40V, preferably 30V；Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for 0.2ng/mL's Leucineenkephalin (leucine enkephalin) calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da ([M+H]⁺) or 554.2615Da ([M-H]^-)。

When separation detection body fluid sample, preferably every 5 samples, which are inserted into 1 blank, prevents cross contamination, can also be inserted into one A Quality Control sample carries out quality control.

Preferably sample room temperature is kept for 0-4 DEG C during separation detection.

According to the method for the present invention, in step 4,

Metabolism group data processing related software is preferably used using data processing software to the data of step 3 separation detection Processing for statistical analysis；

The analysis and processing method includes alignment of data, and peak extracts, and is analyzed using Partial Least Squares OPLS-DA, with P≤ 0.05, CV≤30%, VIP > 1.0, maximum variation multiple >=1.2 filter out potential source biomolecule marker as screening threshold value.Such as Data are analyzed and processed using data processing arts software Pro genesis QI, obtain potential source biomolecule marker.

Wherein the selection of maximum variation multiple considers that fatigue is a kind of recoverable physiological change, and the setting of this numerical value is not It is same as being set as 2.0 even up to 3.0 or 4.0 in disease research, is arranged to compared with fractional value be 1.2.

Accurate molecular weight preferably by liquid chromatography-mass spectrography detection potential source biomolecule marker speculates its molecular formula, utilizes The chromatographic characterization and mass spectral characteristic that chromatographic retention is showed, in conjunction with HMDB (www.hmdb.ca), METLIN (Metlin.scripps.edu), human urine group in one or more databases in Chemspider, MZedDB or KEGG Divided data library is compared, and filters out potential source biomolecule marker, and mass deviation is set as 5ppm.

Above-mentioned marker generation is carried out preferably by MetaboAnalyst online database (www.MetaboAnalyst.ca) It thanks to access inquiry, and is compared with standard items；Metabolic pathway is related to sleep quality, and in liquid chromatograph mass spectrography Instrument detection is consistent with standard items retention time and mass number measurement result, can be used as degree of fatigue associated biomarkers.

Preferably, reappeared in multiple degree of fatigue groups and variation tendency be consistent, but in HMDB (www.hmdb.ca) and The unknown potential source biomolecule marker not retrieved in METLIN (Metlin.scripps.edu) database can be made by purifying Standby sterling out, and the mass spectrum of sterling, ultraviolet, infrared and/or nuclear magnetic data are acquired, Structural Identification is carried out, as degree of fatigue phase Close biomarker.

According to the method for the present invention, in step 5,

The degree of fatigue marker is selected from following one or more kinds of:

(1) urocanic acid (Urocanic acid)；

(2) acetylcytosine (N4-Acetylcytidine)；

(3) 5-hydroxyryptophan (5-Hydroxy-L-tryptophan)；

(4) dimethylguanosine (N2, N2-Dimehtylguanosine)；

(5) antifebrin (N-Acetylarylamine)；With

(6)Alpha-CEHC。

According to the present invention, when the potential degree of fatigue marker filtered out is urocanic acid, that is, it is light to can determine whether that human body occurs Degree fatigue；Preferably, when the potential degree of fatigue marker filtered out and 1.4 times of the up-regulation of its content, it can determine whether that human body goes out Now slight fatigue.

According to the present invention, when the potential degree of fatigue marker filtered out is urocanic acid, acetylcytosine, 5- hydroxyl color ammonia When sour, it can determine whether that moderate fatigue occurs in human body；Preferably, when filter out potential degree of fatigue marker urocanic acid, acetyl born of the same parents Pyrimidine, 5-hydroxyryptophan content decline when, can determine whether human body occur moderate fatigue；It is further preferred that potential tired when filtering out When labor degree marker is urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin and Alpha-CEHC, It can determine whether that moderate fatigue occurs in human body；It is highly preferred that when the potential degree of fatigue marker urocanic acid, acetyl born of the same parents that filter out are phonetic Pyridine, 5-hydroxyryptophan, dimethylguanosine and the decline of antifebrin content, and when the raising of Alpha-CEHC content, it can determine whether human body There is moderate fatigue.

An embodiment of detection method according to the present invention, the urine specimen collected when crewmember's work 2-4 is small When testing result shows that urocanic acid content raises 1.4 times, i.e., degree of fatigue sample is relative to urocanic acid content in non-tired sample When raising 1.4 times, it can determine whether that slight fatigue occurs in crewmember；

An embodiment of detection method according to the present invention, the urine specimen collected when crewmember's work 4-6 is small Testing result show degree of fatigue sample relative in non-tired sample, urocanic acid, acetylcytosine, 5-hydroxyryptophan content When decline multiple is respectively 1.4 or more, 1.3 or more, 1.4 or more, it can determine whether that moderate fatigue occurs in crewmember；

An embodiment of detection method according to the present invention, the urine collected when crewmember working time 5-8 is small Pattern detection as the result is shown degree of fatigue sample relative in non-tired sample, urocanic acid, acetylcytosine, 5-hydroxyryptophan, Dimethylguanosine and antifebrin content decline multiple be respectively 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 with On, and Alpha-CEHC content increases multiple when reaching 1.3 or more, can determine whether that moderate fatigue occurs in crewmember.

The present invention also provides a kind of workload assessment or pre-job check and evaluation whether can be on duty or hilllock in detect For find tired risk and in time adjust or be on duty after check and evaluation post workload method, which is characterized in that including Following steps:

1) when the degree of fatigue sample collected in crewmember's work hilllock 2-4 little Shi judges teams and groups using detection method as above When slight fatigue occurs in member, for the safety in production such as driver, controller sensitive keys post, it is proposed that crewmember first leaves the post Rest (preferably rest 20-30 minutes) is started to work again；

If 2) the degree of fatigue sample collected behind quitting time hilllock is looked in urine specimen using detection method as above Urocanic acid, acetylcytosine, 5-hydroxyryptophan out, and urocanic acid, acetylcytosine, 5-hydroxyryptophan are relative to non-tired sample In downward multiple when being respectively 1.4 or less, 1.3 or less, 1.4 or less, illustrate that the workload of crewmember is appropriate；

3) if work 5 hours or more the degree of fatigue samples collected are detected in urine specimen using detection method as above Urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin and Alpha-CEHC out, and urocanic acid, acetyl Cytimidine, 5-hydroxyryptophan, dimethylguanosine, antifebrin relative to the downward multiple of non-tired sample be respectively 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increases multiple when reaching 1.3 or more, then class Group membership's workload reaches suggest coming off duty at full capacity rest or reduction workload.

The present invention also provides a kind of methods of system assessment of arranging an order according to class and grade, which is characterized in that utilizes as above detection degree of fatigue The result of method judged,

A1) when crewmember work 2-4 it is small when the degree of fatigue sample collected using detection method as above judge teams and groups at There is slight fatigue in member, for security sensitive post, illustrates that the crewmember needs to rest；

The degree of fatigue sample collected when crewmember's work 2-4 is small judges crewmember not using detection method as above When there is slight fatigue, illustrate that the workload of the crewmember that arranges an order according to class and grade is appropriate；Or,

A2) for needing the security sensitive post of significant attention to work, it is small less than 5 that task is executed in crewmember When, urocanic acid, acetylcytosine, 5-hydroxyryptophan biomarker, and degree of fatigue sample are filtered out in the tired sample of collection When this is respectively 1.4 or more, 1.3 or more, 1.4 or more relative to the downward multiple of non-tired sample, there is moderate fatigue, say The bright Workforce Management is unreasonable, it is proposed that stops working, leaves the post the rest long period, or comes off duty or increase personnel's reduction work and bear Lotus；Illustrate crewmember's workload excess load；

If filtering out urocanic acid, acetylcytosine, 5-hydroxyryptophan in the tired sample that the day shift quitting time collects, And degree of fatigue sample is when being respectively 1.4 or less, 1.3 or less, 1.4 or less relative to the downward multiple of non-tired sample, explanation The workload of crewmember is appropriate；Or,

A3 it) for needing the security sensitive post of significant attention to work, works 5-7 hours, receives in crewmember Filtered out in the degree of fatigue sample of collection urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin and Alpha-CEHC, and degree of fatigue sample is respectively 1.4 or more, 1.3 or more, 1.4 relative to the downward multiple of non-tired sample Above, 1.4 or more, 1.4 or more, and Alpha-CEHC content increases multiple when reaching 1.3 or more, illustrates that crewmember occurs Moderate fatigue, crewmember's workload excess load, it is proposed that stop working, leave the post the rest long period, or next or increase personnel Workload is reduced, illustrates crewmember's workload excess load；When 8 hours dutys, it is desirable to reduce the working time reduces work Make load, reduces Safety Risk in Production；Or,

A4) work is 8 hours full, filters out urocanic acid, acetylcytosine, 5- hydroxyl color ammonia in the degree of fatigue sample of collection Acid, dimethylguanosine, antifebrin and Alpha-CEHC, and downward multiple point of the degree of fatigue sample relative to non-tired sample Not Wei 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increase multiple reach 1.3 When above, illustrate that moderate fatigue occurs in crewmember, the Workforce Management employee work is at full capacity.

Technical solution of the present invention has the following beneficial effects:

1) present invention judges that the method for degree of fatigue is more scientific, reasonable.Biochemical processes for tired human body, people Although attempted multiple compounds metabolism and tired correlation research from multi-angles such as operating pressure, sleep, biological rhythms, so And not finding any body metabolism compound yet has correlation with fatigue.The present invention is in view of people are for the people of fatigue Body biochemical process is totally unknown, in selective mechanisms potential source biomolecule marker, using the chromatographic column of opposed polarity, carries out to sample Complete detection and analysis, prevent missing inspection, to improve the accuracy of this method.

2) repeated detection is carried out to same sample using the chromatographic column of a variety of opposed polarities due to the present invention, it on the one hand can be with Avoid missing inspection, on the other hand can be detected simultaneously according to different chromatographic columns the result of same composition to the detection method of chromatographic column into Row verifying.A certain chromatographic column is avoided error occur, to improve the accuracy of screening technique.

3) prior art small molecular metabolism group is mainly used in diagnosis, treatment and medicament research and development of human body diseases etc. Using there is not yet being applied in the research of human normal physiological change.Fatigue is that human body copes with workload, pressure is born A kind of physiological change of lotus, and/or biological rhythm variation and generation, with load intensity and rhythm and pace of moving things variation degree degree of fatigue Change therewith.The present invention in view of Human Physiology variation be itself it is adjustable and by rest as sleep can be able to restore A kind of variation, therefore physiological change causes metabolite level change that will be different from human pathology caused by disease or drug toxicology Lead to the change of metabolite level, i.e. physiological change causes metabolite level to change change caused by being less than pathological change.This hair The bright bottom threshold by metabolin variation multiple is set as 1.2 times, less than 2.0 be normally set up in disease and toxicologic study Times or bigger numerical value.Therefore, detection error caused by screening technique of the invention can avoid because of disease or other factors, thus side The accuracy of method is higher.

4) for the screening technique of this method by the way of tired cross-reference, this mode can deduct feed diet, ring The interference that the factors such as border introduce, while avoiding since fatigue defines ambiguity and error inconsistent and introduce.

5) the degree of fatigue marker and its changes of contents that this method is obtained based on many experiments basis, can be to a small amount of samples This such as 5-10 person-times of sample is detected, is analyzed, being judged the degree of fatigue of crewmember, is provided technology for safety in production and is protected Barrier.

6) result of the present invention for the first time based on many experiments basis goes to judge crewmember's degree of fatigue, by degree of fatigue Judgement, which is realized, can quantify to detect.Inventor has found that degree of fatigue and potential job safety executive capability in industrial production are related therefore tired Labor degree detecting can provide strong new technology and new tool to production safety risk management and control.Utilize the detection knot of degree of fatigue Fruit carries out workload assessment, system of arranging an order according to class and grade assessment in production, and whether pre-job check and evaluation can be on duty, detect and use in hilllock It adjusts in the tired risk of discovery and in time, the method for rear check and evaluation post workload on duty is more scientific, reasonable.

Detailed description of the invention

Fig. 1 is that the polarity HILIC column positive ion mode detection polar compound of drive simulating volunteer's plasma sample is typical total Ion flow graph.

Fig. 2 is that drive simulating volunteer's plasma sample HILIC column positive ion detection polar compound data principal component analysis obtains Component.

Fig. 3 is drive simulating volunteer's plasma sample low pole C₁₈It is typical total that column positive ion mode detects polar lipid component Ion flow graph.

Fig. 4 is drive simulating volunteer's plasma sample low pole C₁₈Column positive ion mode detects polar lipid component data Principal component analysis shot chart.

Fig. 5 is the low pole C of drive simulating volunteer's plasma sample₁₈It is typical that column negative ion mode detects polar lipid component Total ion current figure.

Fig. 6 is drive simulating volunteer's plasma sample low pole C₁₈Column negative ion mode detects polar lipid component data master Constituent analysis shot chart.

Fig. 7 is the low pole C of drive simulating volunteer's plasma sample₁₈Column positive ion mode detects non-polar lipid component allusion quotation Type total ion current figure.

Fig. 8 is drive simulating volunteer's plasma sample low pole C₁₈Column positive ion mode detects non-polar lipid component data Principal component analysis shot chart.

Fig. 9 is the polarity HILIC column positive ion mode detection polar compound of test case drive simulating volunteer's urine specimen Typical total ion current figure.

Figure 10 is test case drive simulating volunteer's urine specimen HILIC column positive ion detection polar compound data principal component Analyze shot chart.

Figure 11 is the low pole C of test case drive simulating volunteer's urine specimen₁₈Column positive ion mode detects low pole group Divide typical total ion current figure.

Figure 12 is test case drive simulating volunteer's urine specimen low pole C₁₈Column positive ion mode detects weakly polar component data Principal component analysis shot chart.

Figure 13 is the low pole C of test case drive simulating volunteer's urine specimen₁₈Column negative ion mode detects weakly polar component allusion quotation Type total ion current figure.

Figure 14 is test case drive simulating volunteer's urine specimen low pole C₁₈Column negative ion mode detects weakly polar component data Principal component analysis shot chart.

Figure 15 is the nonpolar PFPP column positive ion mode detection non-polar component of test case drive simulating volunteer's urine specimen Typical total ion current figure.

Figure 16 is test case drive simulating volunteer's urine specimen nonpolarity PFPP column positive ion mode detection non-polar component number According to principal component analysis shot chart.

Figure 17 is the nonpolar PFPP column negative ion mode detection non-polar component of test case drive simulating volunteer's urine specimen Typical total ion current figure.

Figure 18 is test case drive simulating volunteer's urine specimen nonpolarity PFPP column negative ion mode detection non-polar component number According to principal component analysis shot chart.

Figure 19 is the Structural Identification of test case urocanic acid.

Specific embodiment

Further detailed description is done to method of the invention below in conjunction with specific embodiment.It should be appreciated that following Embodiment is merely illustrative the ground description and interpretation present invention, and is not necessarily to be construed as limiting the scope of the invention.All bases In the range of the technology that above content of the present invention is realized is encompassed by the present invention is directed to protect.

Unless otherwise indicated, raw materials and reagents used in the following embodiment are commercial goods, or can be by Perception method preparation.

Test case 1 simulates the screening that civil aviaton's aircraft driver operates degree of fatigue biomarker in 3.5 hours body fluid Method

1.1 volunteer recruitings and sample collection:

Recruit 99 people of volunteer, enter a group condition: health, no medication, the age at 20-55 years old, wherein 55 people of male, female Property 44 people, female volunteers need not in the menstrual period.

It arranges volunteer to carry out simulation aircraft driver behavior, operates continuously 3.5 hours, induce slight fatigue.

The acquisition of blood and urine specimen: it carries out acquiring blood and urine sample respectively before and after drive simulating operation in volunteer This, blood sample is no less than 2.0mL venous blood using the acquisition of anticoagulant blood-collecting pipe, and urine specimen collects urine simultaneously using Sterilized urine cup It is sub-packed in sterile tube and saves.

Blood of human body, urine specimen save, and blood is centrifuged 3-5min through 4000-5000rpm, upper plasma is sub-packed in dry Net sterile centrifugation tube is stored in -80 DEG C；Urine specimen is sub-packed in sterile centrifugation tube and is stored in -80 DEG C.

The pretreatment of 1.2 human plasmas and urine specimen:

(1) pre-treatment step of plasma sample are as follows: by plasma sample 4 DEG C thawing 30-60 minutes；It is divided into 2 parts, first Part takes 100 μ L blood plasma into the 1.5mL centrifuge tube for having marked label, and the acetonitrile of 300 μ L is added；Sufficiently oscillation 15 seconds, carries out egg White precipitating, 12000rpm, are centrifuged 10 minutes, take 100 μ L of upper solution, be placed in 200 μ L internal lining pipes, for detecting blood plasma by 4 DEG C In polar compound；Second part takes 100 μ L blood plasma into the 1.5mL centrifuge tube for having marked label, and three chloromethanes of 500 μ L are added Alkane/methanol (3:1) solution；Sufficiently oscillation 15 seconds, carries out albumen precipitation, and 12000rpm, is centrifuged 5 minutes, takes lower layer's solution by 4 DEG C 200 μ L, are placed in 1.5mL centrifuge tube；Traditional vacuum is concentrated sample 4 hours, and 300 μ L isopropyls are added into the sample after drying Alcohol/acetonitrile (1:1), concussion mix 40 seconds.12000rpm is centrifuged 5 minutes, is taken 100 μ L of supernatant, is placed in 200 μ L internal lining pipes, For detecting the lipid composition in blood plasma.(2) urine specimen pre-treatment step are as follows: urine specimen is taken out before analysis detection and is placed To room temperature, at 4 DEG C, 12000 rpm are centrifuged 5 minutes, take 100 μ L of supernatant that the dilution of 100 μ L water is added.

1.3 carry out analysis detection to blood plasma and urine specimen using sewage sludge method:

(1) polarity chromatographic column HILIC, low pole chromatographic column C is respectively adopted in the detection of plasma sample₁₈To the polarity in blood Component and lipid composition carry out separation detection respectively.

Using the liquid-phase condition of polarity HILIC chromatographic column are as follows: UPLC BEH Amide HILIC column (2.1 mm × 100mm, 1.7 μm), mobile phase is that A phase composition is the acetonitrile solution containing 0.1% formic acid and 1.0mM ammonium acetate, and B phase composition is Aqueous solution containing 0.1% formic acid and 1.0mM ammonium acetate；40 DEG C of column temperature；Flow rate of mobile phase 0.3mL/min；1.0 μ L of sample volume. Mass Spectrometry Conditions are as follows: the detection of cation ionization mode, mass range are set as 50-1200m/z full scan mode.Electron spray ion Changing the best capillary voltage of device is 3000V (cation) or 2200V (anion), and cone voltage is 30V.Dry gas is nitrogen, Desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C. Use concentration be the leucineenkephalin (leucine enkephalin) of 0.2ng/mL as mass number calibrate internal standard, calibrate from Protonatomic mass is 556.2771Da ([M+H]⁺)。

Using low pole C₁₈The liquid-phase condition of chromatography post detection blood plasma lipide non-polar component are as follows: UPLC CSH C₁₈Column (2.1mm × 100mm, 1.7 μm), mobile phase A phase composition be the acetonitrile containing 0.1% formic acid and 10mM ammonium acetate/ Water is the solution of 6:4, and B phase composition is the solution that acetonitrile/isopropanol containing 0.1% formic acid and 10mM ammonium acetate is 9:1；Column 50 DEG C of temperature；Flow rate of mobile phase 0.3mL/min；1.0 μ L of sample volume.Mass Spectrometry Conditions are as follows: the detection of cation ionization mode, quality model It encloses and is set as 50-1200m/z full scan mode.The best capillary voltage of electro-spray ionization device is 3000V, and cone voltage is 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, Ion source temperature is 100 DEG C.Using concentration is the leucineenkephalin (leucine enkephalin) of 0.2ng/mL as matter It measures number and calibrates internal standard, calibration mass of ion is 556.2771Da ([M+H]⁺)

Using low pole C₁₈The liquid-phase condition of chromatography post detection blood plasma lipide polar compound are as follows: UPLC CSH C₁₈ Column (2.1mm × 100mm, 1.7 μm), mobile phase A phase composition be the aqueous solution containing 0.1% formic acid, B phase composition be containing The methanol solution of 0.1% formic acid.Mass Spectrometry Conditions are as follows: the two kinds of ionization mode detections of cation and anion, mass range setting For 50-1200m/z full scan mode.The best capillary voltage of electro-spray ionization device is 3000V (cation) or 2200V is (negative Ion), cone voltage is 30 V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.It goes molten Agent temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for leucineenkephalin (the bright ammonia of 0.2ng/mL Sour enkephalins) as mass number calibration internal standard, calibration mass of ion is 556.2771Da ([M+H]⁺) or 554.2615Da ([M- H]^-)。

Each plasma sample is all made of 2 kinds of chromatographic columns and detects polarity therein and lipid polarity and non-polar component respectively, Wherein polar compound only detects positive ion mode, and lipid composition detects cation and negative ion mode, i.e., each plasma sample inspection It surveys 4 times, to detect metabolic compounds whole in blood plasma as far as possible.Every 10 samples are inserted into a blank and prevent in detection Cross contamination, and be inserted into a Quality Control sample and carry out quality control.Sample room temperature is kept for 4 DEG C in analyte detection process.

(2) polarity chromatographic column HILIC, low pole chromatographic column C is respectively adopted in the detection of urine specimen₁₈With nonpolar chromatographic column PFPP carries out separation detection to polarity, low pole and the non-polar component in urine respectively.

Using the liquid-phase condition of polarity HILIC chromatographic column are as follows: UPLC BEH Amide HILIC column (2.1 mm × 100mm, 1.7 μm), it is 95% acetonitrile and 5% aqueous solution containing 0.1% formic acid that mobile phase, which is A phase composition, B phase composition be containing There is the aqueous solution of 0.1% formic acid；Column temperature is 40 DEG C, sample volume 2.0 μ L, flow rate of mobile phase 0.3mL/min.Mass Spectrometry Conditions are as follows: just The detection of ion ionization mode, mass range are set as 50-1200m/z full scan mode.The best capillary of electro-spray ionization device Tube voltage is 3000V, and cone voltage is 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for 0.2ng/mL's Leucineenkephalin (leucine enkephalin) calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da ([M+H]⁺)。

Using low pole C₁₈The liquid-phase condition of chromatographic column are as follows: UPLC CSH C₁₈column(2.1mm×100 mm,1.7μ M), mobile phase A phase composition is 95% acetonitrile and 5% aqueous solution containing 0.1% formic acid, and B phase composition is to contain 0.1% formic acid Aqueous solution；Column temperature is 40 DEG C, sample volume 2.0 μ L, flow rate of mobile phase 0.3mL/min.Mass Spectrometry Conditions are as follows: cation and anion Two kinds of ionization mode detections, mass range are set as 50-1200m/z full scan mode.The best capillary of electro-spray ionization device Tube voltage is 3000V (cation) or 2200V (anion), and cone voltage is 30V.Dry gas is nitrogen, desolvation flow velocity For 800L/h, cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for The leucineenkephalin (leucine enkephalin) of 0.2ng/mL calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da([M+H]⁺) or 554.2615Da ([M-H]^-)。

Using the liquid-phase condition of nonpolar PFPP chromatographic column are as follows: UPLC HSS PFPP column (2.1mm × 100mm, 1.7 μm), mobile phase is that A phase composition is the aqueous solution containing 0.1% formic acid, and B phase composition is methanol；Column temperature is 40 DEG C, sample volume 2.0 μ L, flow rate of mobile phase 0.3mL/min.Mass Spectrometry Conditions are as follows: the two kinds of ionization mode detections of cation and anion, quality model It encloses and is set as 50-1200m/z full scan mode.The best capillary voltage of electro-spray ionization device be 3000V (cation) or 2200V (anion), cone voltage are 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for 0.2ng/mL's Leucineenkephalin (leucine enkephalin) calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da ([M+H]⁺) or 554.2615Da ([M-H]^-)。

Each urine specimen is all made of 3 kinds of chromatographic columns and detects polarity, low pole and non-polar component therein respectively, wherein Polar compound only detects positive ion mode, low pole and non-polar component detection cation and negative ion mode, i.e., each urine Pattern detection 5 times, to detect metabolic compounds whole in urine as far as possible.Every 10 samples are inserted into a sky in detection It is white to prevent cross contamination, and be inserted into a Quality Control sample and carry out quality control.Sample room temperature is kept for 4 DEG C in analyte detection process.

1.4 data processings and preliminary screening go out biomarker

The detection data that 435GB is obtained using liquid chromatograph-mass spectrometer analysis detection sample, using metabolism group number According to processing professional software Progenesis QI processing for statistical analysis, including alignment of data, peak is extracted, and packet mode is with mould Quasi- aircraft drives as a kind of processing mode for inducing slight fatigue, pairing grouping is carried out to sample before and after the processing, using inclined Least square method OPLS-DA fractional analysis, using P≤0.05, CV≤30%, VIP > 1.0, maximum multiple >=1.2 that change as sieve Threshold value is selected, preliminary screening goes out potential source biomolecule marker.Wherein the selection of maximum variation multiple considers that fatigue is that one kind can be restored Physiological change, the setting of this numerical value is different from being set as 2.0 even up to 3.0 or 4.0 in disease research, be arranged to compared with decimal Value is 1.2.

Plasma sample detection data is handled according to above-mentioned steps, is detecting blood plasma using polarity HILIC column positive ion mode 8625 compounds of middle polar compound utilize low pole C₁₈Column positive ion mode detects 19646 chemical combination of polar compound in blood plasma 8625 compounds of object and non-polar lipid component utilize low pole C₁₈Column negative ion mode detects polar lipid component in blood plasma In the data of 13648 compounds, fail to filter out qualified biomarker.

Urine specimen detection data is handled according to above-mentioned steps, is utilizing low pole C₁₈Column positive ion mode detects in urine In the data of 12282 compounds of weakly polar component, it is potential source biomolecule marker that Analysis and Screening, which goes out a kind of compound,；Utilizing pole Property HILIC column positive ion mode detection urine in 9490 compounds of polar compound, utilize low pole C₁₈The inspection of column negative ion mode It surveys 24363 compounds of weakly polar component in urine, utilize nonpolar group in nonpolar PFPP column positive ion mode detection urine Divide 13054 compounds and utilizes 7702 compounds of non-polar component in nonpolarity PFPP column negative ion mode detection urine In data, fail to filter out qualified biomarker.

The retrieval of 1.5 biomarker metabolic pathways and structural confirmation

1 kind of potential source biomolecule marker that preliminary screening goes out in step 1.4 is in C₁₈Column retention time are as follows: 3.0 min, accurately Mass number m/z is 139.0508.5ppm is set by mass deviation, utilizes HMDB (www.hmdb.ca) database retrieval ratio It is right, thus it is speculated that the possible structure of the potential source biomolecule marker is C₆H₆N₂O₂And compound name urocanic acid (Urocanic acid).Further purchase urocanic acid sterling is confirmed on liquid chromatograph-mass spectrometer, is added and is compared by standard items, Standard items are consistent with untested compound retention time, and it is consistent to measure accurate mass number, so that it is determined that the potential source biomolecule marker is Urocanic acid.

Urocanic acid metabolic pathway is carried out using MetaboAnalyst online database (www.MetaboAnalyst.ca) to look into It askes, the results showed that urocanic acid belongs to histamine metabolic pathway, further retrieves histamine and sleep correlation, the results showed that histamine and people Somatic sleep is highly relevant.

Urocanic acid changes of contents before and after drive simulating operation is calculated, it is found that its content raises 1.4 times.

It follows that: urocanic acid content raises 1.4 times, i.e. urocanic acid in histamine metabolic pathway when human body is slightly tired The raising of content can reflect human body and be in slight fatigue state.Due to being detected on urocanic acid when human body slight fatigue occurs 1.4 times are adjusted, therefore urocanic acid is raised 1.4 or more to characterize slight tired appearance by us.

The screening technique of tired biomarker in 2 air traffic control person's body fluid of test case

2.1 volunteer recruitings and sample collection:

Volunteer recruiting: 45 people of air traffic control person volunteer is recruited in certain International airport at home, enters a group condition: Health, male, no medication the age 20-35 years old, are divided into 2 groups, first group of (ATC1) 20 people, second group of (ATC2) 25 people. Recruiting 23 people of airport administration and logistics personnel volunteer simultaneously is control group (CON), enters a group condition: health, male, no clothes Medicine, the age 20-35 years old.

Air traffic control person, the air traffic air route that takes charge are boarded a plane safe operation, are generally just referred to Aircraft is waved, manages all aircrafts in entire airspace as a whole with reference to information such as radars.Controller often commands simultaneously at work The operation of multi-aircraft needs the abilities such as energy concentration, accurate judgement and emergency processing, therefore is very easy to fatigue, civil aviation authority Relevant regulations clearly every controller is no more than 2 hours in seat commander's aircraft maximum duration.It is main that fatigue is generated in its work Caused by being mental labour.

This research carries out screening study mainly for day shift controller's degree of fatigue associated biomarkers.By volunteer Day shift is engaged in air traffic control work as a kind of processing for inducing moderate fatigue.

The acquisition of urine specimen: urine specimen of pre-job acquisition is gone to work as non-fatigue in day shift controller volunteer Sample acquires a urine specimen as moderate fatigue sample after controller volunteer's day shift is laid off before leaving offices；Equally, for Also class front and back acquires 2 urine specimens to administration and logistics personnel volunteer respectively on it.Using administration and logistics personnel as control sample This, excludes the interference of human normal physiological metabolism.

ATC1 group sample collection is winter in December, 2014, ATC2 and CON group sample collection is the autumn in October, 2016 Ji Jinhang.

Urine specimen is collected urine and is sub-packed in sterile tube and saved using Sterilized urine cup.

Urine specimen is sub-packed in sterile centrifugation tube and is stored in -80 DEG C.

The pretreatment of 2.2 urine specimens:

Urine specimen pre-treatment step are as follows: by urine specimen taking-up placement to room temperature before analysis detection, at 4 DEG C, 12000rpm is centrifuged 5 minutes, takes 100 μ L of supernatant that the dilution of 100 μ L water is added.

2.3 carry out analysis detection to urine specimen using sewage sludge method:

Polarity chromatographic column HILIC, low pole chromatographic column C is respectively adopted in the detection of urine specimen₁₈With nonpolar chromatographic column PFPP carries out separation detection to polarity, low pole and the non-polar component in urine respectively.

Using low pole C₁₈The liquid-phase condition of chromatographic column are as follows: UPLC CSH C₁₈column(2.1mm×100 mm,1.7μ M), mobile phase A phase composition contains the aqueous solution of 0.1% formic acid, and B phase composition is acetonitrile；Column temperature is 40 DEG C, 2.0 μ L of sample volume, stream Dynamic phase flow velocity 0.3mL/min.Mass Spectrometry Conditions are as follows: the two kinds of ionization mode detections of cation and anion, mass range are set as 50-1200m/z full scan mode.The best capillary voltage of electro-spray ionization device be 3000V (cation) or 2200V (bear from Son), cone voltage is 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Remove solvent Changing temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for the leucineenkephalin (leucine of 0.2ng/mL Enkephalins) as mass number calibration internal standard, calibration mass of ion is 556.2771Da ([M+H]⁺) or 554.2615Da ([M- H]^-)。

2.4 data processings and preliminary screening go out biomarker

The detection data that 261GB is obtained using liquid chromatography/mass spectrometry combined instrument analysis detection sample, using metabolism group number According to processing professional software Progenesis QI processing for statistical analysis, including alignment of data, peak is extracted, and packet mode is to hold Row task is grouped sample before and after the processing as a kind of processing mode for inducing moderate fatigue, use minimum two partially Multiplication OPLS-DA fractional analysis, using P≤0.05, CV≤30%, VIP > 1.0, maximum multiple >=1.2 that change as screening threshold Value, preliminary screening go out potential source biomolecule marker.Wherein the selection of maximum variation multiple considers that fatigue is a kind of recoverable life Reason variation, the setting of this numerical value are different from being set as 2.0 even up to 3.0 or 4.0 in disease research, be arranged to be compared with fractional value 1.2。

First group of controller's ATC1 urine specimen detection data (referring to table 2) is handled according to above-mentioned steps, is utilizing polarity HILIC column positive ion mode detects in urine in the data of 9490 compounds of polar compound, and Analysis and Screening goes out 5 kinds of compounds and is Potential source biomolecule marker；Utilizing low pole C₁₈Column positive ion mode detects 10809 compounds of weakly polar component in urine In data, it is potential source biomolecule marker that Analysis and Screening, which goes out 12 kinds of compounds,；Utilize low pole C₁₈Column negative ion mode detects urine In the data of middle 5247 compounds of weakly polar component, fails Analysis and Screening and go out qualified potential source biomolecule marker；In benefit With in the data of 11414 compounds of non-polar component, Analysis and Screening goes out 6 in nonpolar PFPP column positive ion mode detection urine Kind compound is potential source biomolecule marker；The non-polar component 6176 in using nonpolarity PFPP column negative ion mode detection urine In the data of a compound, it is potential source biomolecule marker that Analysis and Screening, which goes out 2 kinds of compounds,.

Second group of controller's ATC2 urine specimen detection data (referring to table 3) is handled according to above-mentioned steps, is utilizing polarity HILIC column positive ion mode detects in urine in the data of 7903 compounds of polar compound, and Analysis and Screening goes out 4 kinds of compounds and is Potential source biomolecule marker；Utilizing low pole C₁₈Column positive ion mode detects 10463 compounds of weakly polar component in urine In data, it is potential source biomolecule marker that Analysis and Screening, which goes out 5 kinds of compounds,；Utilize low pole C₁₈Column negative ion mode detects in urine In the data of 6014 compounds of weakly polar component, it is potential source biomolecule marker that Analysis and Screening, which goes out a kind of compound,；Utilizing non-pole Property PFPP column positive ion mode detection urine in 9739 compounds of non-polar component data in, Analysis and Screening goes out 4 kinds of chemical combination Object is potential source biomolecule marker；Non-polar component 5996 changes in using nonpolarity PFPP column negative ion mode detection urine In the data for closing object, fails Analysis and Screening and go out qualified potential source biomolecule marker.

Control group CON group urine specimen detection data (referring to table 4) is handled according to above-mentioned steps, is utilizing polarity HILIC Column positive ion mode detects in urine in the data of 8315 compounds of polar compound, and it is potential that Analysis and Screening, which goes out 10 kinds of compounds, Biomarker；Utilizing low pole C₁₈Column positive ion mode detects the data of 10645 compounds of weakly polar component in urine In, it is potential source biomolecule marker that Analysis and Screening, which goes out 17 kinds of compounds,；Utilize low pole C₁₈Column negative ion mode detects weak in urine In the data of 6966 compounds of polar compound, it is potential source biomolecule marker that Analysis and Screening, which goes out 7 kinds of compounds,；Utilizing nonpolarity PFPP column positive ion mode detects in urine in the data of 10318 compounds of non-polar component, and Analysis and Screening goes out 10 kinds of chemical combination Object is potential source biomolecule marker；Non-polar component 6571 changes in using nonpolarity PFPP column negative ion mode detection urine In the data for closing object, fails Analysis and Screening and go out qualified potential source biomolecule marker.

The potential degree of fatigue associated biomarkers filtered out in 2 first groups of controller's urine specimens of table

The potential degree of fatigue associated biomarkers filtered out in 3 second groups of controller's urine specimens of table

The associated biomarkers filtered out in 4. control group urine specimen of table

The retrieval of 2.5 biomarker metabolic pathways and structural confirmation

Compare the potential degree of fatigue associated biomarkers filtered out in three groups of urine specimens, the results showed that (referring to table 5) 3 kinds of potential degree of fatigue associated biomarkers urocanic acids, 5-hydroxyryptophan, acetylcytosines, occur at three groups, this It is outer that there are also 3 kinds of potential degree of fatigue associated biomarkers dimethylguanosines, antifebrin and Alpha-CEHC only in controller Group occurs.

By above-mentioned 6 kinds of compounds at MetaboAnalyst database retrieval (table 4), search result shows compound diformazan Base guanosine belongs to tyrosine metabolic pathway, and antifebrin belongs to aromatic amine metabolic pathway, and 5-hydroxyryptophan belongs to tryptophan metabolism Access, urocanic acid belong to histidine metabolic pathway, and alpha-CEHC and acetylcytosine do not retrieve corresponding metabolic pathway.Into One step literature query the result shows that, above-mentioned 4 retrieved metabolic pathway is related with the sleep of people.

The above results show compared with administration and logistics personnel (control group), by darg, air traffic controller (tired group) increases 3 and is opened with related metabolic pathway of sleeping.

The degree of fatigue associated biomarkers filtered out in 5 urine of table

Test case 3 simulates the screening side that civil aviaton's aircraft driver operates degree of fatigue biomarker in 1 hour body fluid Method

3.1 volunteer recruitings and sample collection:

Recruit 20 people of volunteer, enter a group condition: health, no medication, the age at 20-40 years old, wherein 10 people of male, female Property 10 people, female volunteers need not in the menstrual period.

It arranges volunteer to carry out simulation aircraft driver behavior, operates continuously 1 hour, do not occur feeling of fatigue.

The acquisition of urine specimen: urine specimen is acquired before and after volunteer carries out drive simulating operation, urine specimen uses Sterilized urine cup is collected urine and is sub-packed in sterile tube and saves.

Human urine Sample preservation, urine specimen are sub-packed in sterile centrifugation tube and are stored in -80 DEG C.

The pretreatment of 2.2 human urine samples:

2.3 carry out analysis detection to urine specimen using sewage sludge method:

Polarity chromatographic column HILIC, low pole chromatographic column C is respectively adopted₁₈With nonpolar chromatographic column PFPP to the pole in urine Property, low pole and non-polar component carry out separation detection respectively.

Using low pole C₁₈The liquid-phase condition of chromatographic column are as follows: UPLC CSH C₁₈column(2.1mm×100 mm,1.7μ M), mobile phase A phase composition is the aqueous solution containing 0.1% formic acid, and B phase composition is acetonitrile；Column temperature is 40 DEG C, 2.0 μ L of sample volume, Flow rate of mobile phase 0.3mL/min.Mass Spectrometry Conditions are as follows: the two kinds of ionization mode detections of cation and anion, mass range setting For 50-1200m/z full scan mode.The best capillary voltage of electro-spray ionization device is 3000V (cation) or 2200V is (negative Ion), cone voltage is 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.It goes molten Agent temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for leucineenkephalin (the bright ammonia of 0.2ng/mL Sour enkephalins) as mass number calibration internal standard, calibration mass of ion is 556.2771Da ([M+H]⁺) or 554.2615Da ([M- H]^-)。

1.4 data processings and preliminary screening go out biomarker

The detection data that 35GB is obtained using liquid chromatograph-mass spectrometer analysis detection sample, using metabolism group number According to processing professional software Progenesis QI processing for statistical analysis, including alignment of data, peak is extracted, and packet mode is with mould Quasi- aircraft drives as a kind of processing mode for inducing slight fatigue, pairing grouping is carried out to sample before and after the processing, using inclined Least square method OPLS-DA fractional analysis, using P≤0.05, CV≤30%, VIP > 1.0, maximum multiple >=1.2 that change as sieve Threshold value is selected, preliminary screening goes out potential source biomolecule marker.Wherein the selection of maximum variation multiple considers that fatigue is that one kind can be restored Physiological change, the setting of this numerical value is different from being set as 2.0 even up to 3.0 or 4.0 in disease research, be arranged to compared with decimal Value is 1.2.

Urine specimen detection data is handled according to above-mentioned steps, is detecting blood plasma using polarity HILIC column positive ion mode 4847 compounds of middle polar compound utilize low pole C₁₈Column positive ion mode detects 6916 chemical combination of weakly polar component in urine Object utilizes low pole C₁₈Column negative ion mode detects in blood plasma in the data of 4012 compounds of polar lipid component, and utilization is non- Polarity PFPP column positive ion mode detects 9221 compounds of non-polar component in urine, utilizes nonpolar PFPP column anion mould Formula detects 5074 compounds of non-polar component in urine, fails to filter out qualified biomarker.

By above-mentioned 3 test case results it is found that not filtering out qualified life when tested personnel do not feel fatigue Object marker；Tested person works (drive simulating) 3.5 hours, urocanic acid is expressed as biomarker, and it contains Amount raises 1.4 times relative to non-tired sample；Extending with the working time has, and more biomarkers are detected, such as are worked After the lighter work in administrative personnel's day shift 8 hours of load, three kinds of urocanic acid, 5HTP, acetylcytosine biological markers Object expresses and lowers 1.4,1.4,1.3 times respectively；Controller's day shift 8 big for workload, security responsibility risk is high is small When work after, then six kinds of biomarker expressions, wherein urocanic acid, 5HTP, acetylcytosine, dimethylguanosine, Antifebrin, Alpha-CEHC, preceding 5 kinds of downwards multiple is respectively 1.4,1.3,1.4,1.4,1.4, and Alpha-CEHC contains Amount increases multiple and reaches 1.3.

The testing result and situation of change such as the following table 6 of 6 kinds of biomarkers:

6 degree of fatigue associated biomarkers of table

1 pair of simulation aircraft driver teams and groups, civil aviaton of embodiment carry out degree of fatigue detection

1.1 volunteer recruitings and sample collection:

The pretreatment of 1.2 human urine samples:

1.3 carry out analysis detection to urine specimen using sewage sludge method:

1.4 data processings and preliminary screening go out biomarker

The detection data that 435GB is obtained using liquid chromatograph-mass spectrometer analysis detection sample, using metabolism group number According to processing professional software Progenesis QI processing for statistical analysis, including alignment of data, peak extraction is driven with simulating aircraft Sail as a kind of processing mode for inducing slight fatigue, analyzed using Partial Least Squares OPLS-DA, with P≤0.05, CV≤ 30%, as screening threshold value, preliminary screening goes out potential source biomolecule marker for VIP > 1.0, maximum variation multiple >=1.2.It is wherein maximum The selection of variation multiple is a kind of recoverable physiological change in view of fatigue, and the setting of this numerical value is different from setting in disease research 2.0 even up to 3.0 or 4.0 are set to, is arranged to compared with fractional value be 1.2.

The retrieval of 1.5 biomarker metabolic pathways and structural confirmation

1 kind of potential source biomolecule marker that preliminary screening goes out in 1.4 steps is in C₁₈Column retention time are as follows: 3.0 min, accurately Mass number m/z is 139.0508.5ppm is set by mass deviation, retention time deviation is set as 0.5min, with 6 degree of fatigue of table Biomarker compares potential source biomolecule marker chromatography retention behavior and mass number is consistent with urocanic acid.Further use urocanic acid Standard items are added by standard items and are compared, and standard items are consistent with untested compound retention time, and it is consistent to measure accurate mass number, So that it is determined that the potential source biomolecule marker is urocanic acid.

Compare urocanic acid variation multiple to raise before and after drive simulating and reach 1.48 times.Then the simulation civil aviaton aircraft drives There is slight fatigue in member crewmember.Be on duty after rest 20-30 minutes operation again it is recommended that crewmember leaves the post.

The degree of fatigue of 2 air traffic control person teams and groups of embodiment detects

2.1 volunteer recruitings and sample collection:

Volunteer recruiting: 25 people of air traffic control person volunteer is recruited in certain International airport at home, enters a group condition: Health, male, no medication, the age 20-35 years old.Air traffic control person flies on the air traffic air route that takes charge Machine safe operation, is generally exactly to command aircraft, manages all aircrafts in entire airspace as a whole with reference to information such as radars.? Controller often commands the operation of multi-aircraft simultaneously in work, needs the abilities such as energy concentration, accurate judgement and emergency processing, Therefore it is very easy to fatigue, civil aviation authority's relevant regulations clearly every controller is small no more than 2 in seat commander's aircraft maximum duration When.It is mainly caused by mental labour that fatigue is generated in its work.

The acquisition of urine specimen: urine specimen of pre-job acquisition is gone to work as non-fatigue in day shift controller volunteer Sample acquires a urine specimen as moderate fatigue sample after controller volunteer's day shift is laid off before leaving offices.Sample collection It is to be carried out autumn in October, 2016, controller teams and groups reach the day shift working time 8 hours.

Urine specimen is collected urine and is sub-packed in sterile tube and saved at -80 DEG C using Sterilized urine cup.

The pretreatment of 2.2 urine specimens:

2.3 carry out analysis detection to urine specimen using sewage sludge method:

Using the liquid-phase condition of polarity HILIC chromatographic column are as follows: UPLC BEH Amide HILIC column (2.1 mm × 100mm, 1.7 μm), it is 95% acetonitrile and 5% aqueous solution containing 0.1% formic acid that mobile phase, which is A phase composition, B phase composition be containing There is the aqueous solution of 0.1% formic acid；Column temperature is 40 DEG C, sample volume 2.0 μ L, flow rate of mobile phase 0.3mL/min.Mass Spectrometry Conditions are as follows: just The detection of ion ionization mode, mass range are set as 50-1200m/z full scan mode.The best capillary of electro-spray ionization device Tube voltage is 3000V, and cone voltage is 30V.Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30 L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for 0.2ng/mL's Leucineenkephalin (leucine enkephalin) calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da ([M+H]⁺)。

2.4 data processings and preliminary screening go out biomarker

The detection data that 65GB is obtained using liquid chromatography/mass spectrometry combined instrument analysis detection sample, using metabolism group number According to processing professional software Progenesis QI processing for statistical analysis, including alignment of data, peak is extracted, to sample before and after the processing This is analyzed, and is analyzed using Partial Least Squares OPLS-DA, with P≤0.05, CV≤30%, VIP > 1.0, maximum variation times Number >=1.2 goes out potential source biomolecule marker as screening threshold value, preliminary screening.Wherein the selection of maximum variation multiple is in view of fatigue It is a kind of recoverable physiological change, the setting of this numerical value is different from being set as 2.0 even up to 3.0 or 4.0 in disease research, It is arranged to compared with fractional value be 1.2.

According to above-mentioned steps, processing controller organizes urine specimen detection data (referring to table 7), is utilizing polarity HILIC column Positive ion mode detects in urine in the data of 7903 compounds of polar compound, and it is potential life that Analysis and Screening, which goes out 4 kinds of compounds, Object marker；Utilizing low pole C₁₈Column positive ion mode detects in urine in the data of 10463 compounds of weakly polar component, It is potential source biomolecule marker that Analysis and Screening, which goes out 5 kinds of compounds,；Utilize low pole C₁₈Column negative ion mode detects low pole in urine In the data of 6014 compounds of component, it is potential source biomolecule marker that Analysis and Screening, which goes out a kind of compound,；Utilizing nonpolarity PFPP Column positive ion mode detects in urine in the data of 9739 compounds of non-polar component, and it is latent that Analysis and Screening, which goes out 4 kinds of compounds, In biomarker；The number of 5996 compounds of non-polar component in using nonpolarity PFPP column negative ion mode detection urine In, fails Analysis and Screening and go out qualified potential source biomolecule marker.

7 controller of table organizes the potential tired associated biomarkers filtered out in urine specimen

The retrieval of 2.5 biomarker metabolic pathways and structural confirmation

14 kinds of potential source biomolecule markers that preliminary screening goes out in 2.4 steps, pass through chromatographic column, retention time and accurate matter Number is measured, sets 5ppm for mass deviation, retention time deviation is set as 0.5min, with 6 degree of fatigue biomarker ratio of table Compared with preceding 6 kinds of potential source biomolecule markers and the in table 6 the 2nd to the 7th urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethyl bird Glycosides, antifebrin are consistent with the information of Alpha-CEHC, are confirmed.By urocanic acid standard items add compare, standard items with Untested compound retention time is consistent, in addition, it is consistent to measure accurate molecular weight through mass spectrum, so that it is determined that wherein the first potential life Object marker is urocanic acid.

Further relatively 6 kinds of biomarkers change multiple, urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethyl bird Glycosides, antifebrin lower 1.44,1.38,1.62,1.5,1.33 times of 1.78, Alpha-CEHC up-regulation respectively.

Conclusion, the controller teams and groups day shift work 8 hours, moderate fatigue occur.

More than, embodiments of the present invention are illustrated.But the present invention is not limited to above embodiment.It is all Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention Within the scope of shield.

Claims

1. a kind of method based on human urine detection teams and groups' degree of fatigue, which is characterized in that described method includes following steps:

The urine specimen of step 1. acquisition crewmember；

The sample that step 2. acquires step 1 pre-processes；

The potential source biomolecule marker that step 4 filters out is compared step 5. with degree of fatigue marker, determines to be detected class The degree of fatigue of group membership.

2. the method as described in claim 1, which is characterized in that in step 1:

The urine specimen derives from the crewmember of health, when the crewmember is women, preferably not in menstruation The women of phase；

Preferably, the acquisition step includes acquiring non-tired sample and degree of fatigue sample；

Preferably, the acquisition step of sample includes: to acquire it before crewmember goes to work work or before simulated operation induction fatigue Urine specimen is as non-tired sample；According to degree of fatigue, collected after crewmember's work or after simulated operation induction fatigue Its urine specimen is as degree of fatigue sample；

Preferably, the phases such as stream time length, work position, workload and duty arrangement of each member in the teams and groups It is same or similar；

Preferably, the teams and groups that the teams and groups can for example be constituted for driver, controller, flight-line maintenance personnel or healthcare givers Deng；

Preferably, the teams and groups can be the sample size group of 5 people or more, such as the sample size group of 8-20 people, such as the sample of 8-10 people This amount group；

Preferably, the sample collection procedure of step 1 further includes that sample is being not higher than -20 DEG C of items after having acquired urine specimen Cryo-conservation is carried out under part；Such as urine specimen is sub-packed in sterile centrifugation tube and is stored in -80 DEG C；

Preferably, appropriate antibiotic can also be added into sample during cryo-conservation, avoid causing part generation because of bacterium Thank to the decomposition of object；Such as the sodium azide that mass concentration is 0.05-0.1% is added into sample.

3. method according to claim 1 or 2, which is characterized in that in step 2:

Pre-treatment step includes: to be centrifuged urine specimen, and taking supernatant and being optionally diluted to supernatant is suitable for subsequent step The aqueous solution of rapid analysis detection concentration, such as sample is diluted using the water of 1-3 times of volume；For example, by using following steps pair Urine specimen is pre-processed: being taken out urine specimen before analysis detection and is placed to room temperature, at 4 DEG C, 12000rpm is centrifuged 5 points Clock takes 100 μ L of supernatant that the dilution of 100 μ L water is added.

4. the method according to claim 1, which is characterized in that in step 3:

The liquid chromatography-mass spectrometry is preferably sewage sludge method；

Preferably, the detection sample is the degree of fatigue sample and non-tired sample of teams and groups；

Preferably, the liquid chromatogram uses the preferred HILIC column of polarity chromatographic column, the preferred C of low pole chromatographic column₁₈Column and nonpolarity The preferred PFPP column of chromatographic column carries out separation detection to polarity, low pole and the non-polar component in urine respectively；

Preferably, when the liquid chromatogram uses polarity chromatographic column separation detection polar compound, mass spectrography is preferably using detection Positive ion mode；When the liquid chromatogram is using low pole and non-polar column separation detection low pole and non-polar component, matter Spectrometry uses cation and anion both of which；

Preferably, using the liquid-phase condition of polarity HILIC chromatographic column are as follows: UPLC BEH Amide HILIC column [(2.1- 4.6)mm╳(100-250)mm,1.5-5μm]；

Preferably, mobile phase is that A phase composition is volume fraction 95-100% acetonitrile or methanol and volume fraction is that 0-5% contains The aqueous solution of 0.1-0.5% formic acid or acetic acid or trifluoroacetic acid, B phase composition are to contain 0.1-0.5% formic acid or acetic acid or trifluoro The aqueous solution of acetic acid；

Preferably, column temperature is 20-40 DEG C, sample volume 0.5-5.0 μ L, flow rate of mobile phase 0.3-1.0mL/min；

Preferably, using low pole C₁₈The liquid-phase condition of chromatographic column are as follows: UPLC CSH C₁₈Column [(2.1-4.6) mm ╳ (100- 250)mm,1.5-5μm)]；

Preferably, mobile phase A phase composition is the aqueous solution containing 0.1-5% formic acid or acetic acid or trifluoroacetic acid, and B phase composition is second Nitrile or methanol；

Preferably, liquid chromatography uses the liquid-phase condition of nonpolarity PFPP chromatographic column are as follows: UPLC HSS PFPP column [(2.1- 4.6)mm╳(100-250)mm,1.5-5μm)]；

Preferably, it is the aqueous solution containing 0.1-5% formic acid or acetic acid or trifluoroacetic acid that mobile phase, which is A phase composition, and B phase composition is Methanol or acetonitrile；

Preferably, column temperature is 20-40 DEG C, sample volume 0.3-5.0 μ L, flow rate of mobile phase 0.3-1.0mL/min；

Preferably, the testing conditions of the mass spectrography are as follows:

When being detected using cation ionization mode, mass range is set as 50-1200m/z full scan mode.Electron spray from Sonization device capillary voltage is 2500-3500, and preferably 3000V, cone voltage is 20-40V, preferably 30V.Dry gas is nitrogen, Desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C. Use concentration be the leucineenkephalin (leucine enkephalin) of 0.2ng/mL as mass number calibrate internal standard, calibrate from Protonatomic mass is 556.2771Da ([M+H]⁺)；

Preferably, when being detected using two kinds of ionization modes of cation and anion, mass range is set as 50-1200m/z Full scan mode；Electro-spray ionization device capillary voltage be 2000-3500, preferably 3000V (cation) or 2200V (bear from Son), cone voltage is 20-40V, preferably 30V；Dry gas is nitrogen, and desolvation flow velocity is 800L/h, and cone gas velocity is 30L/h.Desolvation temperature is 400 DEG C, and ion source temperature is 100 DEG C.Use concentration for 0.2ng/mL's Leucineenkephalin (leucine enkephalin) calibrates internal standard as mass number, and calibration mass of ion is 556.2771Da ([M +H]⁺) or 554.2615Da ([M-H]^-)；

Preferably, when separation detection body fluid sample, every 5 samples, which are inserted into 1 blank, prevents cross contamination, can also be inserted into one Quality Control sample carries out quality control；

Preferably, sample room temperature is kept for 0-4 DEG C during separation detection.

5. method according to any of claims 1-4, which is characterized in that in the step 4:

Metabolism group data processing related software is preferably used to carry out using data processing software the data of step 3 separation detection Statistical analysis processing；

Preferably, the analysis and processing method includes alignment of data, and peak extracts, and is analyzed using Partial Least Squares OPLS-DA, with P≤0.05, CV≤30%, VIP > 1.0, maximum variation multiple >=1.2 filter out potential source biomolecule marker as screening threshold value. Such as data are analyzed and processed using data processing arts software Pro genesis QI, obtain potential source biomolecule marker.

Preferably, its molecular formula is speculated using the accurate molecular weight of liquid chromatography-mass spectrography detection potential source biomolecule marker, utilize color The chromatographic characterization and mass spectral characteristic that spectrum retention time is showed, in conjunction with HMDB (www.hmdb.ca), METLIN (Metlin.scripps.edu), human urine group in one or more databases in Chemspider, MZedDB or KEGG Divided data library is compared, and filters out potential source biomolecule marker, and mass deviation is set as 5ppm；

Preferably, above-mentioned marker generation is carried out using MetaboAnalyst online database (www.MetaboAnalyst.ca) It thanks to access inquiry, and is compared with standard items；Metabolic pathway is related to sleep quality, and in liquid chromatograph mass spectrography Instrument detection is consistent with standard items retention time and mass number measurement result, can be used as degree of fatigue associated biomarkers；

Preferably, it is reappeared in multiple degree of fatigue groups and variation tendency is consistent, but in HMDB (www.hmdb.ca) and METLIN (Metlin.scripps.edu) the unknown potential source biomolecule marker not retrieved in database can be prepared pure by purifying Product, and the mass spectrum of sterling, ultraviolet, infrared and/or nuclear magnetic data are acquired, Structural Identification is carried out, as degree of fatigue associated biomolecule Marker.

6. the method according to claim 1 to 5, which is characterized in that in step 5:

The degree of fatigue marker is selected from following one or more kinds of:

(1) urocanic acid (Urocanic acid)；

(2) acetylcytosine (N4-Acetylcytidine)；

(3) 5-hydroxyryptophan (5-Hydroxy-L-tryptophan)；

(4) dimethylguanosine (N2, N2-Dimehtylguanosine)；

(5) antifebrin (N-Acetylarylamine)；With

(6)Alpha-CEHC。

7. as the method according to claim 1 to 6, which is characterized in that in step 5:

When the potential degree of fatigue marker filtered out be urocanic acid when, and its content raise 1.4 times when, can determine whether that human body goes out Now slight fatigue；

Preferably, when the potential degree of fatigue marker filtered out is urocanic acid, acetylcytosine, 5-hydroxyryptophan, can sentence There is moderate fatigue in disconnected human body；

Preferably, when the content decline of the potential degree of fatigue marker urocanic acid, acetylcytosine, 5-hydroxyryptophan that filter out When, it can determine whether that moderate fatigue occurs in human body；

It is further preferred that when the potential degree of fatigue marker filtered out is urocanic acid, acetylcytosine, 5-hydroxyryptophan, diformazan When base guanosine, antifebrin and Alpha-CEHC, it can determine whether that moderate fatigue occurs in human body；

It is highly preferred that when the potential degree of fatigue marker urocanic acid, the acetylcytosine, 5-hydroxyryptophan, dimethyl that filter out Guanosine and the decline of antifebrin content, and when the raising of Alpha-CEHC content, it can determine whether that moderate fatigue occurs in human body.

8. the method according to claim 1 to 7, which is characterized in that in step 5:

It is when the urine specimen testing result collected when crewmember's work 2-4 is small shows that urocanic acid content raises 1.4 times, i.e., tired When labor degree sample raises 1.4 times relative to urocanic acid content in non-tired sample, it can determine whether that slight fatigue occurs in crewmember；

Preferably, when crewmember work 4-6 it is small when the urine specimen testing result collected show degree of fatigue sample relative to In non-fatigue sample, urocanic acid, acetylcytosine, 5-hydroxyryptophan content decline multiple be respectively 1.4 or more, 1.3 or more, When 1.4 or more, it can determine whether that moderate fatigue occurs in crewmember；

Preferably, the urine specimen testing result collected when crewmember working time 5-8 is small shows degree of fatigue sample phase For in non-tired sample, urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine and the decline of antifebrin content are again Number is respectively 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increases multiple and reaches When to 1.3 or more, it can determine whether that moderate fatigue occurs in crewmember.

9. a kind of workload assessment or pre-job check and evaluation whether can be on duty or hilllock in detection for finding tired wind The method of check and evaluation post workload after adjusting nearly and in time or being on duty, which comprises the steps of:

1) when the degree of fatigue sample collected in crewmember's work hilllock 2-4 little Shi is utilized as described in claim any one of 1-8 When method judges that slight fatigue occurs in crewmember, for the safety in production such as driver, controller sensitive keys post, it is proposed that class Group membership first leave the post rest (preferably rest 20-30 minutes) start to work again；

If 2) the degree of fatigue sample collected behind quitting time hilllock utilizes any one of claim 1-8 the method such as urinating Find out urocanic acid, acetylcytosine, 5-hydroxyryptophan in liquid sample, and urocanic acid, acetylcytosine, 5-hydroxyryptophan relative to When downward multiple in non-fatigue sample is respectively 1.4 or less, 1.3 or less, 1.4 or less, illustrate that the workload of crewmember is suitable When；

3) if work 5 hours or more the degree of fatigue samples collected utilize any one of claim 1-8 the method such as urinating Urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin and Alpha-CEHC are detected in liquid sample, and The downward multiple point of urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin relative to non-tired sample Not Wei 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increase multiple reach 1.3 When above, then crewmember's workload reaches at full capacity, it is proposed that come off duty rest or reduction workload.

10. a kind of method for system assessment of arranging an order according to class and grade, which is characterized in that the method according to claim 1 is utilized,

A1) the degree of fatigue sample collected when crewmember's work 2-4 is small judges that crewmember goes out using detection method as above Now when slight fatigue, for security sensitive post, illustrate that the crewmember needs to rest；

The degree of fatigue sample collected when crewmember's work 2-4 is small judges that crewmember does not occur using detection method as above When slight fatigue, illustrate that the workload of the crewmember that arranges an order according to class and grade is appropriate；Or,

A2) for needing the security sensitive post of significant attention to work, in crewmember's execution task less than 5 hours, Urocanic acid, acetylcytosine, 5-hydroxyryptophan biomarker, and degree of fatigue sample phase are filtered out in the tired sample of collection When being respectively 1.4 or more, 1.3 or more, 1.4 or more for the downward multiple of non-tired sample, there is moderate fatigue, illustrate the row Class's system is unreasonable, it is proposed that stops working, leaves the post the rest long period, or comes off duty or increase personnel and reduce workload；Explanation Crewmember's workload excess load；

If filtering out urocanic acid, acetylcytosine, 5-hydroxyryptophan in the tired sample that the day shift quitting time collects, and tired When labor degree sample is respectively 1.4 or less, 1.3 or less, 1.4 or less relative to the downward multiple of non-tired sample, illustrate teams and groups The workload of member is appropriate；Or,

A3 it) for needing the security sensitive post of significant attention to work, works 5-7 hours in crewmember, collection Urocanic acid, acetylcytosine, 5-hydroxyryptophan, dimethylguanosine, antifebrin and Alpha- are filtered out in degree of fatigue sample CEHC, and degree of fatigue sample relative to the downward multiple of non-tired sample is respectively 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increases multiple when reaching 1.3 or more, illustrates that moderate occurs in crewmember Fatigue, crewmember's workload excess load, it is proposed that stop working, leave the post the rest long period, or come off duty or increase personnel and reduce Workload；When 8 hours dutys, it is desirable to reduce the working time reduces workload, reduces Safety Risk in Production；Or,

A4) work is 8 hours full, filters out urocanic acid, acetylcytosine, 5-hydroxyryptophan, two in the degree of fatigue sample of collection Methylguanosine, antifebrin and Alpha-CEHC, and degree of fatigue sample is respectively relative to the downward multiple of non-tired sample 1.4 or more, 1.3 or more, 1.4 or more, 1.4 or more, 1.4 or more, and Alpha-CEHC content increases multiple and reaches 1.3 or more When, illustrate that moderate fatigue occurs in crewmember, Workforce Management crewmember works at full capacity.