CN110007044A - The quantitative detecting method of a variety of organic acids in a kind of human urine - Google Patents
The quantitative detecting method of a variety of organic acids in a kind of human urine Download PDFInfo
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- CN110007044A CN110007044A CN201910410353.1A CN201910410353A CN110007044A CN 110007044 A CN110007044 A CN 110007044A CN 201910410353 A CN201910410353 A CN 201910410353A CN 110007044 A CN110007044 A CN 110007044A
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Abstract
The present invention relates to biochemistry detection technical fields, and in particular to a kind of method of a variety of organic acid contents in synchronous quantitative detection human urine.The detection method first mixes human urine with internal standard solution, then by target organic acid extraction purification in such a way that organic solvent extracts, then liquid chromatogram separation is used to synchronize qualitative and quantitative analysis to target organic acid with method associated with the detection of mass spectrum primary and secondary ion pair after purification with strong anion exchange column.Abstraction purification step ensure that being fully dissolved out for target organic acid in urine, exclude the interference of impurity in urine, it is ensured that the Stability and veracity of testing result.The synchronization batch detection of this method organic acid of structured sort multiplicity suitable for human urine has many advantages, such as that high sensitivity, precision are high, reproducible, detection speed is fast.Sufficiently meet the needs of clinical examination, clinical value with higher.
Description
Technical field
The present invention relates to a variety of organic in biochemistry detection technical field more particularly to a kind of synchronous quantitative detection human urine
The method of acid content.
Background technique
Organic acid is a major class compound of generation during body analytic metabolism.They come from dietary protein, fat
And carbohydrate, by body for generating cellular energy and providing nutrition necessary to cell function.The test of urine organic acid
The metabolin of selection can be used as the important diagnostic index of abnormal metabolism.Metabolic imbalance is a kind of common and universal disease, can
It can be the basis of many chronic diseases, such as fatigue, gastrointestinal dysfunction, muscle/joint prob, emotional handicap and headache.This
A little diseases usually have resistance to long-term treatment and lasting improvement.Organic acid analysis traditionally can be used for early detection/exclusion
Or monitoring dysbolism.Urine specimen can be used for assessing enteron aisle, liver and nervous system health and energetic supersession and nutrition lacks
It is weary.
Protein, fat and carbohydrate are used to cellular energy as the main food of human body and provide thin
Nutrition necessary to born of the same parents' function.It but simultaneously also should include other necessary nutrients in food, only in these necessary nutrition
Food just can be converted into energy with the help of element and nutrition is absorbed by the body.When food metabolism, certain essential nutrients are lacked
It is weary to will lead to that energy generates and the access of nutrient absorption is obstructed, while the organic acid metabolite generated will be discharged into urine.
Therefore individual energetic supersession situation can be assessed by the measurement of organic acid in urine and whether specific nutrient lacks.For
Many organic acids of measurement, abnormal high level generally indicates that specific metabolic pathway is obstructed in urine, which must
The nutrient level needed is lower.The test of urine organic acid helps to understand the executive mode of nutrient metabolic, and determines metabolism week
Phase, there may be unbalanced places.For example, B family vitamin, especially B1 (thiamine), B3 (niacin) and B5 are (general
Acid), required confactor is provided for the energy pathway of all body cells.When food is metabolized, vitamin B is being needed to participate in
The step of in form specific compound.These steps occur in carbohydrate metabolism, wherein forming pyruvic acid and lactic acid.This
The auxiliary enzyme activity of a little raised this reaction of explanation of chemical levels is low, it is therefore desirable to increase B family vitamin, especially thiamines
Element and pantothenic acid.After the intake for increasing these essential nutrients, the access of reaction is got through again, the energy generation efficiency of cell
Restore.Almost each link of body system requires enzyme co-factor, these confactors such as vitamin or minerals lack
The weary body function that can be influenced extensively, including immune, endocrine, muscle skeleton and metabolic system.For many organic of measurement
Acid, abnormal high level generally indicates that in urine decompose the compound needed for nutrient level it is lower.On this basis, functional
The demand of specific nutrition element is assessed, creation personalized therapy program is the optimal selection for improving patient symptom.
Therefore, this quantitative detecting method for researching and developing a variety of organic acid metabolites in a kind of urine.This method has
Detection sensitivity is high, reproducible, detection speed is fast, detects advantage of large quantities, can efficiently to organic acid metabolite into
The accurate quantitative detection of row, has good scientific research and commercial application prospect.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of high performance liquid chromatography combination tandem mass spectrometries to detect people
The method of a variety of organic acid metabolites in body urine.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
Step 1, urine pass through organic solvent for target organic acid abstraction purification after mixing with internal standard solution, be dissolved in water after being dried with nitrogen
In, column purification then is exchanged with strong anion, is dried with nitrogen after organic solvent elution, it is soluble in water to obtain sample to be tested；
Step 2 detects above-mentioned sample to be tested by liquid chromatogram and mass spectrometry, obtains the type and content of organic acid.
Further, it includes ethyl acetate, n-hexane, methylene chloride, three that organic solvent used is extracted in above-mentioned steps 1
Chloromethanes and t-butyl methyl ether, preferred organic extraction solvent are ethyl acetate.Further, the organic extraction solvent
For ethyl acetate.
Further, the eluting solvent of strong anion exchange column is methanol or acetone (adding 2% formic acid) in above-mentioned steps 1.
Further, in above-mentioned steps 2 specifically includes the following steps:
Step A: weighing organic acid standard items, dissolved with methanol, obtains the titer of various concentration；
Step B: the internal standard solution after measuring above-mentioned methanol constant volume is separately added into the titer that step A is obtained, uses ultrapure water
It is centrifuged after constant volume, takes supernatant as titer to be measured；
Step C: the sample to be tested that the obtained titer to be measured of step B and step 1 are obtained is detected by LC-MS, is had
The type and content of machine acid metabolic product.
Further, the organic acid metabolite include ethanedioic acid, suberic acid, ethyl malonic acid, lactic acid, pyruvic acid,
Beta-hydroxy-butanoic acid, citric acid, cis-aconitic, isocitric acid, α-ketoglutaric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, α -one-
Beta-methyl valeric acid, succinic acid, fumaric acid, malic acid, Hydroxymethylglutaryl, xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), beta-hydroxy isovaleric acid and methylmalonic acid.
It is produced using the above-mentioned metabolism of organic acids in organic solvent extraction and strong anion exchange column purification process human urine
Object is separated 20 kinds of organic acid metabolites using high performance liquid chromatography, mass spectrum Isotopic Internal Standard sizing technique is recycled, with standard
The concentration of product and internal standard compound ratio is X-axis, and the peak area ratio of standard items and internal standard compound is Y-axis, establishes calibration curve, and calculating above-mentioned has
The content of machine acid metabolic product.
Further, the liquid phase chromatogram condition of above-mentioned steps C LC-MS detection are as follows:
Chromatographic column is bonded-phase chromatography column, and sample volume is 5-50 μ L, and mobile phase includes A and B, and wherein A is aqueous solution, and B is mixing
Solution, the flow velocity of mobile phase are 0.1-2.0mL/min；The gradient (with volume percentage) of mobile phase:
0-1min, A:30-100%, B:70-0%；
1.1-5min, A:10-60%, B:90-60%；
5.1-9min, A:0-50%, B:100-50%；
9.1-11min A:50-100%, B:50-0%；
11.1min, A:30-100%, B:70-0%.
Further, above-mentioned bonded-phase chromatography column is the bonded-phase chromatographies column such as C18 or C8, specification be 50-150mm ×
2.1-4.6mm,1.8-5μm。
Further, in liquid phase chromatogram condition, mobile phase A is the ammonium acetate solution+0.1%-1% of 1-10mmol/L
The pure water of formic acid, Mobile phase B are the mixed solution of acetonitrile methanol, and the mass ratio of acetonitrile and methanol is 9-1:1-9.
Further, the Mass Spectrometry Conditions of above-mentioned LC-MS detection are as follows: ESI ion source, anion MRM scanning, atomization gas
Flow velocity is 5-10L/min, and gas curtain gas velocity is 5-20L/min, ion source voltage 2000-4500V, ion source temperature 200-
400℃。
Further, under Negative electrospray ionization detection pattern, using the scanning of the mass spectrum mode of multiple-reaction monitoring, object
Ion pair monitoring it is as follows: ethanedioic acid (m/z 145.2-83.1), suberic acid (m/z 173.2-111.2), ethyl malonic acid
(m/z 131.1-87.1), lactic acid (m/z 89.1-43.1), pyruvic acid (m/z 87.1-43.1), beta-hydroxy-butanoic acid (m/z
103.1-59.1), citric acid (m/z 191.1-87.1), cis-aconitic (m/z 173.1-85.1), isocitric acid (m/z
191.1-73.1), α-ketoglutaric acid (m/z 145.1-101.1), α-ketoisovaleric acid (m/z 115.1-115.1), α -one are different
Caproic acid (m/z 129.2-129.2), α -one-Beta-methyl valeric acid (m/z 129.2-129.2), succinic acid (m/z 117.1-
73.0), fumaric acid (m/z 115.1-71.0), malic acid (m/z 133.1-115.0), Hydroxymethylglutaryl (m/z
117.1-59.0), xanthurenic acid (4,8-Er Qiangjikuilinjiasuan) (m/z 204.2-160.1), beta-hydroxy isovaleric acid (m/z 117.1-73.1) and methyl-prop two
Sour (m/z 117.1-73.1).Internal standard: D3- methylmalonic acid (m/z 120.1-73.1), D3- ethyl malonic acid (m/z
134.1-87.1), D4- citric acid (m/z 195.1-87.1).
Compared with the prior art, advantage of the invention is that following 3 points.
1, the detection method of organic acid metabolite mixes human urine and internal standard solution in human urine provided by the invention
The analysis that LC-MS is carried out after organic solvent extraction and strong anion column purification is successively carried out after conjunction to mixed liquor.Organic solvent
Target organic acid metabolite can be made to be fully dissolved out and detection efficiency can be improved by strong anion column purification after extraction, from
And the type and content of organic acid metabolite in sample to be tested can be really reacted, so that the accuracy of testing result, again
Renaturation and stability greatly improve.Compared with other sample processing methods, impurity interference is greatly reduced in this detection, peak type pair
Claim, quantitative detection result is more reliable and more stable, and detection error is small between batch, and accuracy rate is high.
2, the detection method of organic acid metabolite is suitable for all metabolism of organic acids in human urine provided by the invention
The synchronization batch detection of product.These organic acid metabolite constituent structure are complicated, while carrying out quick separating identification with very
Big technical difficulty.The method of liquid phase separation and Mass Spectrometer Method provided by the invention does not need to carry out target product at derivative place
Reason can carry out qualitative and quantitative analysis to all target products with efficiently and accurately, meet the needs of high-volume laboratory testing, have
There is higher application value.
3, the detection method of organic acid metabolite has high sensitivity, accuracy in human urine provided by the invention
High, the reproducible and fireballing advantage of detection is created on this basis suitable for the demand of functional assessment specific nutrition element
Building personalized therapy program is the optimal selection for improving patient symptom.
Detailed description of the invention
1: three kind of sample processing method quantitative detection of attached drawing compares.In A-F6 group picture from top to bottom: 1. sample is by organic
Without the direct loading of ion exchange column purification after solvent extraction；2. sample is extracted without organic solvent, pure by ion exchange column
Loading after change；3. sample is by organic solvent extraction and the loading after ion exchange column purification.Extracted through organic solvent and pass through from
The peak type of the sample quantitative detection target product of sub- exchange column after purification is symmetrical, no bifurcated maize phenomenon, quantitative accurate.(A: suitable
Aconitic acid；B: isocitric acid and citric acid；C: α-ketoisovaleric acid；D: α-ketoglutaric acid；E: α -one-Beta-methyl valeric acid and α -one are different
It is sour；F: adipic acid).
Attached drawing 2: the ion flow graph of all 20 kinds of organic acid metabolite quantitative detections in urine specimen.Our standard measure is quasi-
Really, baseline is steady, and resultant error is small between batch, and method stability, accuracy are high.(left column from top to bottom are as follows: ethanedioic acid, pungent
Diacid, ethyl malonic acid, pyruvic acid, lactic acid, beta-hydroxy-butanoic acid, citric acid and isocitric acid, cis-aconitic, isocitric acid,
α-ketoglutaric acid；Right column are from top to bottom are as follows: succinic acid, fumaric acid, malic acid, Hydroxymethylglutaryl, α-ketoisovaleric acid, α -one are different
Caproic acid and α -one-Beta-methyl valeric acid, xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), beta-hydroxy isovaleric acid and methylmalonic acid).
Specific embodiment
According to following embodiments, the present invention can be better understood.As it will be easily appreciated by one skilled in the art that embodiment institute
The content of description is merely to illustrate the present invention, but should not will not limit the present invention described in detail in claims.
Based on the embodiment of the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiments shall fall within the protection scope of the present invention.
Embodiment 1.
The sample of research experiment is medical to 2 months parts from West China Hospital endocrine metabolism section, Sichuan Province in January, 2019
The urine specimen of volunteer.
Sample treatment.
Same urine specimen takes 3 parts, respectively takes 100 μ L.Sample process is carried out respectively.Wherein sample process difference is as follows.
1.: 100 μ L urines are mixed with internal standard solution, be added 100 μ L ethyl acetate extraction, be dried with nitrogen it is rear soluble in water,
Sample introduction after 0.22 μm of membrane filtration.
2.: 100 μ L urines are mixed with internal standard solution, and in the strong anion exchange column after activation is added, methanol (2% formic acid) is washed
It is 2 times, each 1mL de-, amount to 2mL eluent, dry, soluble in water, 0.22 μm of filter of being volatilized under conditions of 60 DEG C of heating with nitrogen evaporator
Sample introduction after film filtering.
3.: 100 μ L urines are mixed with internal standard solution, and the extraction of 100 μ L ethyl acetate is added, is dried with nitrogen rear soluble in water, adds
In strong anion exchange column after entering activation, methanol (2% formic acid) is eluted 2 times, each 1mL, amounts to 2mL eluent, heating 60
Volatilized under conditions of DEG C with nitrogen evaporator dry, soluble in water, sample introduction after 0.22 μm of membrane filtration.
Instrument: the triple level four bars mass spectrographs of API 4000 (Applied Biosystem company)；Agilent liquid chromatogram
System (matches autosampler)；Ten a ten thousandth balances；Chromatographic column (C18)；Nitrogen evaporator；High speed desktop refrigerated centrifuge；Freeze-drying
Machine；Solid-phase extraction column；Adjustable pipette；Glass apparatus, beaker, graduated cylinder etc..
Reagent consumptive material: Chromatographic Pure Methanol；Acetonitrile；Formic acid；Ammonium acetate；Ethyl acetate；Ammonium hydroxide.
Standard items: ethanedioic acid, suberic acid, ethyl malonic acid, lactic acid, pyruvic acid, beta-hydroxy-butanoic acid, citric acid, cis- crow
Head acid, isocitric acid, α-ketoglutaric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, α -one-Beta-methyl valeric acid, succinic acid, fumaric acid,
Malic acid, Hydroxymethylglutaryl, xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), beta-hydroxy isovaleric acid and methylmalonic acid, internal standard D3- methylmalonic acid, D3- ethyl
Malonic acid, D4- citric acid are purchased from Sigma-Aldrich.
Method
Chromatographic condition: mobile phase A: 0.5%v/v formic acid+5mM ammonium acetate solution；Mobile phase B: 0.5%v/v formic acid methanol is molten
Liquid.Column model:, using gradient elution mode.Flow velocity is 0.3mL/min, and column temperature is 45 DEG C, and sample volume is 10 μ L.
Mass Spectrometry Conditions: electrospray ionisation anionic textiles mode, using the scan pattern of multiple-reaction monitoring.Spray voltage is
4kV；Collision gas is；Ion source atomization gas is；Heating auxiliary gas is；The solvent temperature is gone to be.The object of monitoring is as follows: ethanedioic acid
(m/z 145.2-83.1), suberic acid (m/z 173.2-111.2), ethyl malonic acid (m/z 131.1-87.1), lactic acid
(m/z 89.1-43.1), pyruvic acid (m/z 87.1-43.1), beta-hydroxy-butanoic acid (m/z 103.1-59.1), citric acid (m/
Z 191.1-87.1), cis-aconitic (m/z 173.1-85.1), isocitric acid (m/z 191.1-73.1), α -one penta 2
Acid (m/z 145.1-101.1), α-ketoisovaleric acid (m/z 115.1-115.1), α-ketoisocaproic acid (m/z 129.2-
129.2), α -one-Beta-methyl valeric acid (m/z 129.2-129.2), succinic acid (m/z 117.1-73.0), fumaric acid (m/z
115.1-71.0), malic acid (m/z 133.1-115.0), Hydroxymethylglutaryl (m/z 117.1-59.0), xanthurenic acid (4,8-Er Qiangjikuilinjiasuan) (m/
Z 204.2-160.1), beta-hydroxy isovaleric acid (m/z 117.1-73.1) and methylmalonic acid (m/z 117.1-73.1).It is interior
Mark: D3- methylmalonic acid (m/z 120.1-73.1), D3- ethyl malonic acid (m/z 134.1-87.1), D4- citric acid (m/
z 195.1—87.1)。
Cluster voltage is gone to each target respectively, the conditions such as collision voltage carry out system optimization, to reach optimal stabilization
Property and sensitivity.
Standard items are prepared: being accurately weighed standard items respectively from 10mg to 400mg, be respectively placed in the volumetric flask of 10mL, add
Enter methanol solution, is configured to the standard items mother liquor that concentration is respectively 1-100mg/mL；It is pipetted from the above standard items mother liquor respectively
10 μ L are added in same centrifuge tube, add methanol 800ml, and obtaining concentration is 10-1000 μ g/mL hybrid standard product solution, so
Concentration equimultiple is diluted into 6 gradients step by step with methanol afterwards；It is configured into the hybrid standard liquid of gradient.
Internal standard product are weighed, the inner mark solution for being completely dissolved to obtain that concentration is 4mg/mL is added.Then with methanol that internal standard is molten
The concentration dilution of liquid to 100ug/mL, preparation obtains internal standard working solution.
1. sample is after organic solvent extracts without the direct loading of ion exchange column purification；2. sample is without organic solvent
Extraction, passes through loading after ion exchange column purification；3. sample is by organic solvent extraction and the loading after ion exchange column purification.
Data analysis result shows: although 1. sample is extracted by organic solvent, due to pure not by ion exchange column
Change, there are many impurity to interfere in sample.In the position of target product appearance there are the influence of impurity peaks, unstability of base line is caused.Especially
It is that the lower target product peak type of content is asymmetric, quantitative analysis results inaccuracy.2. sample directly passes through ion exchange column
Purifying, but extracted without organic solvent, equally exist a large amount of impurity interference.It shows as target product peak type bifurcated or has packet
Paddy causes quantitative inaccuracy, and error information detection is big between batch.3. sample is extracted by organic solvent extract target production first
Then object is concentrated by ion exchange column purification, exclude a large amount of interfering substances in urine.Referring to attached drawing 1.The peak of target product
Type is symmetrical, no bifurcated maize phenomenon, and quantitative accurate, resultant error is small between batch, and method stability, accuracy are high.
Embodiment 2.
The sample of methodological study experiment is from West China Hospital endocrine metabolism section, Sichuan Province in January, 2019 to 2 months portions
Divide the urine specimen of medical volunteer.
Sample treatment.
The extraction of 100 μ L ethyl acetate is added in 100 μ L urines, is dried with nitrogen rear soluble in water, is then mixed with internal standard solution,
In strong anion column after activation is added, methanol (2% formic acid) is eluted 2 times, each 1mL, amounts to 2mL eluent, heats 60 DEG C
Under conditions of volatilized dry, soluble in water, sample introduction after 0.22 μm of membrane filtration with nitrogen evaporator.
Reagent and instrument and equipment are the same as embodiment 1 in the present embodiment.
Chromatographic condition and Mass Spectrometer Method condition are the same as embodiment 1 in the present embodiment.
Method validation.
1. specificity.
2 parts of 0.5ml bare substrate are taken, point 2 groups of processing:
1. retaining blank；
2. the mixed mark working solution (10-1000ug/ml) that 10 μ l contain 20 organic acid standard items is added, and 10 μ l inner mark solutions
(100ug/ml)
2 groups of samples, which are vortexed, mixes 10s, places 30min.It carries out extracting and crossing column purification later.Sample introduction is analyzed after membrane filtration.
Compare two groups of chromatograms, analyzes endogenous material in urine and test compound and interior target are interfered.
Specificity verification result shows to go out peak position in standard items, and bare substrate sample does not occur significantly interfering with peak.20
The standard items of kind organic acid and the peak type of urine sample are symmetrical, interfere substantially without miscellaneous peak in low concentration.
2. extraction standard curve.
Take series of concentrations extraction standard curve sample, from low concentration to high concentration continuous sample introduction, 2 sample introductions of each concentration into
Row analysis.Using measured object EM concentration as abscissa, measured object and internal standard compound peak area ratio are ordinate, using linear regression,
Obtain regression equation and related coefficient.
Calibration curve: internal standard quanitation is used, using software using the concentration of standard items and internal standard compound ratio as X-axis, standard items
It is Y-axis with internal standard compound peak area ratio, establishes calibration curve, calculates the concentration of determinand in saliva.Each target product is respective
Concentration range in linear fit equation it is linearly good, fitting coefficient be greater than 0.99, meet quantitative requirement.It see the table below.
3. minimum quantitative limit.
Bare substrate 0.5ml is taken, a certain amount of mixed mark working solution (0.125-25ug/ml) and each 10 μ of internal standard working solution is added
L carries out Sample pretreatment.Sample introduction is analyzed, and 6 parts of same concentration parallel processing.Extraction standard curve is substituted into, concentration is calculated
Precision is less than 25%, and when the rate of recovery is between 100 ± 30%, which is minimum quantitative limit.The signal-to-noise ratio of a certain concentration is big
In 3, it is determined as minimum detectability.Experimental result shows, the rate of recovery between 81%-119%, precision between 8%-15%,
Each minimum quantitative limit of organic acid is same as above range of linearity lowest range in table.
4. matrix effect.
Divide 2 groups of processing samples:
1. methanol 0.5ml is taken, it is high, normal, basic to be separately added into mixed mark working solution and internal standard working solution according to 3 concentration of quality-control sample
Each 10 μ l carries out Sample pretreatment.Sample introduction is analyzed；
2. bare substrate 0.5ml is taken, it is high, normal, basic to be separately added into mixed mark working solution and internal standard work according to 3 concentration of quality-control sample
Make each 10 μ l of liquid, carries out Sample pretreatment.Sample introduction is analyzed.Each concentration chooses 3 parts of separate sources bare substrates.
Calculate: the standard items peak area that the 2nd group is obtained respectively divided by the 1st group of resulting respective standard product peak area (×
100%) to get the matrix effect of each standard items.It is required that matrix effect is in 85%-115%, and matrix mark-on and methanol solution add
Mark should all meet RSD < 20%.
The experimental results showed that the matrix effect range of 20 organic acids is 86%-112%, it can satisfy method validation and want
It asks.
5. accuracy and precision.
It selects Quality Control high, normal, basic and LLOQ concentration carries out accuracy and precision is investigated.Sample is prepared using bare substrate.Together
One day every a sample is continuously analyzed 6 times, accuracy of measurement and withinday precision.
Prepare 4 concentration samples again daily, daily continuous sample introduction 2 times measures for three days on end, analyzes day to day precision.Mark
Song need to be measured simultaneously.
The RSD < 15% of precision, accuracy is in 100 ± 25% ranges.The accuracy of LLOQ is loosened to ± 30%,
RSD≤20%.
The experimental results showed that the accuracy of 20 organic acids is between 80%-121%, withinrun precision is in 5%-12%
Between, day to day precision is between 10%-15%.Between 79%-121%, withinrun precision exists for minimum quantitative limit accuracy
Between 9%-13%, day to day precision < 20%.
Stability.
1. day internal stability.
4 DEG C of placements, 24 hours stability.
Senior middle school's low concentration quality-control sample after pre-treatment, is placed 24 hours, difference sample introduction measurement in 0,4,8,24 hour at 4 DEG C,
Every sample measures 2 needles every time.Calculate the rate of recovery and RSD value for measuring concentration.
The time required to standing time should be more than single batched sample analysis；It is required that RSD < 15%, accuracy 100 ±
In 15% range.
The experimental results showed that tested 4 DEG C of 20 kinds of organic acids placements 24 hours are stable.The accuracy model of 4 measurements
It encloses for 85%-111%, RSD is between 10%-14%.
Sample stability test.
Stability test under urine specimen room temperature and 4 DEG C of placements.
Urine specimen is taken, by the method for the invention point after placing 0,24 and 72 hour under the conditions of room temperature and 4 DEG C
The content of all 20 kinds of organic acids is not measured.It is required that accuracy is in 100 ± 25% ranges compared with 0 measured value, it is believed that should
Time point target is stablized.
Sample is placed at room temperature and 4 DEG C 24 hours as the result is shown, and 20 organic acids keep stable.And when in room temperature and
After being placed 72 hours at 4 DEG C, part organic acid such as ethanedioic acid, citric acid, isocitric acid, α-ketoglutaric acid, α-ketoisovaleric acid, α-
Ketoisocaproate, α -one-Beta-methyl valeric acid are not able to satisfy acceptance condition, show more than 72 hours determinands of room temperature and 4 DEG C of placements not
It is able to maintain stabilization.
Stability test under urine specimen freezing conditions.
Urine specimen is taken, after (- 20 DEG C) under freezing conditions are placed 0,3,7,14,30,60,90 day, by the present invention
The method measures the content of all 20 kinds of organic acids.It is required that compared with 0 measured value, accuracy in 100 ± 25% ranges,
Think that the time point target is stablized.
Sample is placed at -20 DEG C in 60 days as the result is shown, and 20 organic acids keep stable.And it is transferred when at -20 DEG C
After setting 90 days, part organic acid for example ethanedioic acid, citric acid, isocitric acid, α-ketoglutaric acid, α-ketoisovaleric acid, α-ketoisocaproic acid,
α -one-Beta-methyl valeric acid, xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), pyruvic acid, succinic acid, fumaric acid etc. is not able to satisfy acceptance condition, shows that -20 DEG C of placements are super
It crosses 90 days determinands and is not able to maintain stabilization.
Embodiment 3.
The sample of experiment is from West China Hospital endocrine metabolism section, Sichuan Province in January, 2019 to the medical aspiration in 2 months parts
The urine specimen of person.
Sample process is the same as embodiment 2 in the present embodiment.
Reagent and instrument and equipment are the same as embodiment 1 in the present embodiment.
Chromatographic condition and Mass Spectrometer Method condition are the same as embodiment 1 in the present embodiment.
Urine sample detects 20 kinds of organic acid ion flow graphs, sees attached drawing 2.
Measurement result and data statistics:
Claims (8)
1. the quantitative detecting method of a variety of organic acids in a kind of human urine, which is characterized in that the detection method includes as follows
Step:
1) by organic solvent by target organic acid abstraction purification after urine is mixed with internal standard solution, be dried with nitrogen it is rear soluble in water,
Then column purification is exchanged with strong anion, is dried with nitrogen after organic solvent elution, it is soluble in water to obtain sample to be tested；
2) above-mentioned sample to be tested is detected by liquid chromatogram and mass spectrometry, is synchronized qualitative to the progress of a variety of organic acids and quantitative
Batch detection.
2. detection method according to claim 1, it is characterised in that extracting organic solvent used in the step includes
Ethyl acetate, n-hexane, methylene chloride, chloroform and t-butyl methyl ether, preferred organic extraction solvent are ethyl acetate.
3. detection method according to claim 1, in the step eluting solvent of strong anion exchange column be methanol or
Acetone (adds 2% formic acid).
4. -3 described in any item detection methods according to claim 1, which is characterized in that the step specifically includes following step
It is rapid:
Step 1) weighs various organic acid standard items, is dissolved to obtain the titer of various concentration with methanol；
It is internal standard solution that step 2), which weighs internal standard sample constant volume, and the titer in step 1) is then added, is centrifuged after ultrapure water constant volume,
Take supernatant as titer to be measured；
Step 3) examines sample to be tested obtained in titer to be measured obtained in step 2) and claim 1 by LC-MS
It surveys, the type and content of organic acid in urine is calculated by standard curve.
5. detection method according to claim 4, which is characterized in that the liquid phase color that LC-MS detects in the step 3)
Spectral condition are as follows: chromatographic column is bonded-phase chromatography column, and sample volume is 5-50 μ L, and mobile phase A is the ammonium acetate water containing 1-10mmol/L
The pure water of solution+0.1%-1% formic acid, Mobile phase B are the mixed solution of acetonitrile methanol, and the mass ratio of acetonitrile and methanol is 9-1:
1-9；The flow velocity of mobile phase is 0.1-2.0mL/min；The gradient (with volume percentage) of mobile phase:
0-1min, A:30-100%, B:70-0%；
1.1-5min, A:10-60%, B:90-60%；
5.1-9min, A:0-50%, B:100-50%；
9.1-11min A:50-100%, B:50-0%；
11.1min, A:30-100%, B:70-0%.
6. detection method according to claim 5, which is characterized in that the bonded-phase chromatography column is the bonding such as C18 or C8
Phase chromatographic column, specification be 50-150mm × 2.1-4.6mm, 1.8-5 μm.
7. detection method according to claim 4, which is characterized in that the mass spectrum item that LC-MS detects in the step 3)
Part are as follows: use electron spray (ESI) ion source, anion MRM scanning mode, atomization gas flow velocity is 5-10L/min, gas curtain gas velocity
For 5-20L/min, ion source voltage is -2000-4500V, and ion source temperature is 200-400 DEG C.
8. detection method according to claim 1-7, which is characterized in that the organic acid compound includes human body
The main organic acid product of generation is metabolized in urine；Using the scanning of the mass spectrum mode of multiple-reaction monitoring, the ion pair of object is supervised
It surveys as follows: ethanedioic acid (m/z 145.2-83.1), suberic acid (m/z 173.2-111.2), ethyl malonic acid (m/z
131.1-87.1), lactic acid (m/z 89.1-43.1), pyruvic acid (m/z 87.1-43.1), beta-hydroxy-butanoic acid (m/z
103.1-59.1), citric acid (m/z 191.1-87.1), cis-aconitic (m/z 173.1-85.1), isocitric acid (m/z
191.1-73.1), α-ketoglutaric acid (m/z 145.1-101.1), α-ketoisovaleric acid (m/z 115.1-115.1), α -one are different
Caproic acid (m/z 129.2-129.2), α -one-Beta-methyl valeric acid (m/z 129.2-129.2), succinic acid (m/z 117.1-
73.0), fumaric acid (m/z 115.1-71.0), malic acid (m/z 133.1-115.0), Hydroxymethylglutaryl (m/z
117.1-59.0), xanthurenic acid (4,8-Er Qiangjikuilinjiasuan) (m/z 204.2-160.1), beta-hydroxy isovaleric acid (m/z 117.1-73.1) and methyl-prop two
Sour (m/z 117.1-73.1)；Internal standard: D3- methylmalonic acid (m/z 120.1-73.1), D3- ethyl malonic acid (m/z
134.1-87.1), D4- citric acid (m/z 195.1-87.1).
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110412157A (en) * | 2019-07-17 | 2019-11-05 | 广州金评检测研究院有限公司 | The measuring method of niacin in a kind of niacinamide raw material |
| CN111638287A (en) * | 2020-06-11 | 2020-09-08 | 成都益康谱科技有限公司 | Quantitative detection method for detecting multiple organic acids in human body dry urine filter paper sheet |
| CN112147253A (en) * | 2020-09-25 | 2020-12-29 | 安徽瑞思威尔科技有限公司 | UPC2-PDA-Q-Tof/MS detection method for 42 effective components in ganoderma lucidum wine |
| CN114778749A (en) * | 2022-05-05 | 2022-07-22 | 杭州度安医学检验实验室有限公司 | Method for efficiently determining 4 organic acids in cell lysate |
| CN115902043A (en) * | 2022-12-30 | 2023-04-04 | 上海药坦药物研究开发有限公司 | Method for determining content of impurities in 8-bromooctanoic acid sample |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763526A (en) * | 2004-10-22 | 2006-04-26 | 上海市徐汇区中心医院 | Method for simultaneously detecting multiple phenolic acid compounds in blood or urine |
| US20100126254A1 (en) * | 2004-12-06 | 2010-05-27 | Applied Biosystems, Llc | Method for Screening Urine for Organic Acids |
| CN102621262A (en) * | 2012-02-28 | 2012-08-01 | 杭州博圣生物技术有限公司 | Method for detecting organic acid in urine by means of gas chromatography and mass spectrography |
| CN103837634A (en) * | 2014-03-21 | 2014-06-04 | 上海硕源健标生物医学科技有限公司 | Method for simultaneously detecting contents of various organic acids in urine of human body |
-
2019
- 2019-05-17 CN CN201910410353.1A patent/CN110007044A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763526A (en) * | 2004-10-22 | 2006-04-26 | 上海市徐汇区中心医院 | Method for simultaneously detecting multiple phenolic acid compounds in blood or urine |
| US20100126254A1 (en) * | 2004-12-06 | 2010-05-27 | Applied Biosystems, Llc | Method for Screening Urine for Organic Acids |
| CN102621262A (en) * | 2012-02-28 | 2012-08-01 | 杭州博圣生物技术有限公司 | Method for detecting organic acid in urine by means of gas chromatography and mass spectrography |
| CN103837634A (en) * | 2014-03-21 | 2014-06-04 | 上海硕源健标生物医学科技有限公司 | Method for simultaneously detecting contents of various organic acids in urine of human body |
Non-Patent Citations (4)
| Title |
|---|
| AIPING LIU ET AL.: "Solid phase extraction procedure for urinary organic acid analysis by gas chromatography mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| ELSEVIER SCIENCE B.V.: "Procedures for MS analysis of clinically relevant compounds", 《CLINICAL MASS SPECTROMETRY》 * |
| IRENE M.L.W.K. ET AL.: "Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism（包括Electronic supplementary material）", 《JOURNAL OF INHERITED METABOLIC DISEASE》 * |
| 高素莲 等: "HPLC法测定人体尿液中有机酸的含量", 《安徽大学学报(自然科学版)》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110412157A (en) * | 2019-07-17 | 2019-11-05 | 广州金评检测研究院有限公司 | The measuring method of niacin in a kind of niacinamide raw material |
| CN111638287A (en) * | 2020-06-11 | 2020-09-08 | 成都益康谱科技有限公司 | Quantitative detection method for detecting multiple organic acids in human body dry urine filter paper sheet |
| CN112147253A (en) * | 2020-09-25 | 2020-12-29 | 安徽瑞思威尔科技有限公司 | UPC2-PDA-Q-Tof/MS detection method for 42 effective components in ganoderma lucidum wine |
| CN112147253B (en) * | 2020-09-25 | 2022-04-01 | 安徽瑞思威尔科技有限公司 | UPC2-PDA-Q-Tof/MS detection method for 42 effective components in ganoderma lucidum wine |
| CN114778749A (en) * | 2022-05-05 | 2022-07-22 | 杭州度安医学检验实验室有限公司 | Method for efficiently determining 4 organic acids in cell lysate |
| CN115902043A (en) * | 2022-12-30 | 2023-04-04 | 上海药坦药物研究开发有限公司 | Method for determining content of impurities in 8-bromooctanoic acid sample |
| CN115902043B (en) * | 2022-12-30 | 2023-08-18 | 上海药坦药物研究开发有限公司 | Method for measuring impurity content in 8-bromooctanoic acid sample |
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