CN107340341A - A kind of TMAO immue quantitative detection reagent box and method - Google Patents
A kind of TMAO immue quantitative detection reagent box and method Download PDFInfo
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- CN107340341A CN107340341A CN201710502265.5A CN201710502265A CN107340341A CN 107340341 A CN107340341 A CN 107340341A CN 201710502265 A CN201710502265 A CN 201710502265A CN 107340341 A CN107340341 A CN 107340341A
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- UYPYRKYUKCHHIB-UHFFFAOYSA-N trimethylamine N-oxide Chemical compound C[N+](C)(C)[O-] UYPYRKYUKCHHIB-UHFFFAOYSA-N 0.000 title claims abstract description 241
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title claims abstract description 25
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims abstract description 146
- 238000012360 testing method Methods 0.000 claims abstract description 77
- 239000007788 liquid Substances 0.000 claims abstract description 75
- 230000003647 oxidation Effects 0.000 claims abstract description 75
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 75
- 150000001875 compounds Chemical class 0.000 claims abstract description 53
- 239000007864 aqueous solution Substances 0.000 claims abstract description 45
- 210000002966 serum Anatomy 0.000 claims abstract description 44
- 108010088751 Albumins Proteins 0.000 claims abstract description 43
- 102000009027 Albumins Human genes 0.000 claims abstract description 43
- 238000010183 spectrum analysis Methods 0.000 claims abstract description 32
- 230000001376 precipitating effect Effects 0.000 claims abstract description 26
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 60
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 34
- 239000012224 working solution Substances 0.000 claims description 23
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 16
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 10
- 150000001412 amines Chemical class 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 235000019253 formic acid Nutrition 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- 239000001301 oxygen Substances 0.000 claims description 8
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 7
- 235000019257 ammonium acetate Nutrition 0.000 claims description 7
- 239000007791 liquid phase Substances 0.000 claims description 7
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000012956 testing procedure Methods 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 150000001768 cations Chemical class 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 2
- 150000002926 oxygen Chemical class 0.000 claims 1
- 239000012460 protein solution Substances 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 abstract description 13
- 239000011159 matrix material Substances 0.000 abstract description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract description 5
- 229940098773 bovine serum albumin Drugs 0.000 abstract description 5
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 93
- 210000002381 plasma Anatomy 0.000 description 68
- 238000010790 dilution Methods 0.000 description 14
- 239000012895 dilution Substances 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000010200 validation analysis Methods 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 239000013078 crystal Substances 0.000 description 5
- 238000010813 internal standard method Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000012496 blank sample Substances 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 238000000132 electrospray ionisation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
A kind of TMAO immue quantitative detection reagent box and method, including step：TMAO is added in the human serum albumins aqueous solution and prepares the series of oxidation trimethylamine titer with concentration gradient；Add internal standard compound and precipitating reagent centrifuges to obtain supernatant, carry out liquid chromatogram and mass spectral analysis respectively, obtain the ratio of the chromatographic peak area of TMAO and internal standard compound in each TMAO titer, and obtain the relation of ratio and TMAO concentration；After test plasma sample is added into internal standard compound and precipitating reagent centrifugation, carry out liquid chromatogram and mass spectral analysis, the ratio of the chromatographic peak area of TMAO and internal standard compound in test plasma sample is obtained, the concentration of TMAO in sample to be tested is obtained according to the relation between the ratio and TMAO concentration.So avoid causing the insecure problem of testing result with blank bio-matrix of the human plasma into phase-splitting difference farther out using water, bovine serum albumin white liquor or phosphate buffer solution etc., improve the reliability of testing result.
Description
Technical field
The present invention relates to a kind of content of material detection technique field, and examination is quantitatively detected more particularly to a kind of TMAO
Agent box and method.
Background technology
The quantitative detecting method of TMAO (Trimetlylamine oxide, TMAO) in blood plasma has important
Clinical value.At present, many methods of the quantitative detection of TMAO contribute to detect the TMAO in aquatic products
Concentration, it is mostly simple chromatography, and sensitivity and the degree of accuracy are limited.And detect the TMAO in the biological specimens such as blood plasma, serum
Concentration, LC-MS technology (HPLC-MS, LC-MS-MS) are methods the most frequently used at present.Almost all people
All contain TMAO in blood plasma, therefore the acquisition of blank bio-matrix turns into difficult point in Method validation, many methods are using water, ox
The blank bio-matrix such as seralbumin liquid, phosphate buffer, due to these blank bio-matrixes and sample to be tested matrix into
Farther out, this causes its Method validation convincingness not high to phase-splitting difference, and obtained testing result reliability is bad.
The content of the invention
Based on this, it is necessary to provide a kind of TMAO quantitative detecting reagent for the reliability that can improve testing result
Box and method.
A kind of TMAO immue quantitative detection reagent box, including series of oxidation trimethylamine titer, internal standard compound and precipitating reagent；
The solute of the series of oxidation trimethylamine titer is TMAO, the solvent behaviour seralbumin aqueous solution.
Above-mentioned TMAO immue quantitative detection reagent box, series of oxidation trimethylamine titer are water-soluble using human serum albumins
Liquid is matrix, so avoid using water, bovine serum albumin white liquor or phosphate buffer etc. and human plasma into phase-splitting difference compared with
Remote blank bio-matrix causes the obtained insecure problem of testing result, and then is advantageous to improve the reliable of testing result
Property.And using the internal standard method of internal standard compound, precipitated by precipitating reagent, be advantageous to further improve the reliability of testing result.
In one of the embodiments, the concentration range of the series of oxidation trimethylamine titer is 2~500ng/mL.
In one of the embodiments, the internal standard compound is deuterated TMAO, and the precipitating reagent is acetonitrile.
In one of the embodiments, the quality of human serum albumins and the body of water in the human serum albumins aqueous solution
Product ratio is (3~10) Kg:100L.
A kind of TMAO quantitative detecting method, using above-mentioned TMAO immue quantitative detection reagent box, including it is following
Step：
Step (1), serial oxygen of the TMAO preparation with concentration gradient is added in the human serum albumins aqueous solution
Change trimethylamine titer；
Step (2), the series of oxidation trimethylamine titer is added into internal standard compound and precipitating reagent centrifuge to obtain supernatant, respectively
Carry out liquid chromatogram and mass spectral analysis, obtain in each TMAO titer the chromatographic peak area of TMAO with it is described interior
Mark the ratio of the chromatographic peak area of thing, and obtain the ratio and the TMAO titer TMAO concentration it
Between relation；
Step (3), the test plasma sample addition internal standard compound and the precipitating reagent are centrifuged to obtain to supernatant, progress liquid phase
Chromatogram and mass spectral analysis, obtain the chromatogram of the chromatographic peak area of TMAO and the internal standard compound in the test plasma sample
The ratio of peak area, and it is dense according to the TMAO of the ratio that step (2) obtains and the TMAO titer
Relation between degree, obtain the concentration of TMAO in the test plasma sample.
Above-mentioned TMAO quantitative detecting method, the human serum albumins aqueous solution is used as matrix, so avoid using
The blank bio-matrix into phase-splitting difference farther out of water, bovine serum albumin white liquor or phosphate buffer etc. and human plasma causes
The insecure problem of testing result arrived, and then be advantageous to improve the reliability of testing result.And internal standard method is used, obtain institute
The relation between the TMAO concentration of ratio and the TMAO titer is stated, is advantageous to further improve detection knot
The reliability of fruit.In addition after internal standard compound and precipitating reagent centrifugation pretreatment are added to plasma sample to be measured, a liquid chromatogram is passed through
Between the obtained ratio of mass spectral analysis and step (2) and the TMAO concentration of the TMAO titer
Relation, the concentration of the TMAO of test plasma sample can be quickly obtained, avoided complicated using long column or liquid-phase condition
Cause the sample analysis time length that method detects, it is impossible to the problem of meeting the requirement of Big Clinical Samples amount quick detection, be blood plasma
Quantifying for TMAO concentration provides a kind of fast and accurately high-sensitivity detecting method in sample.
In one of the embodiments, the step (1) comprises the following steps：TMAO is dissolved in methanol aqueous solution
Prepare the series of oxidation trimethylamine working solution with concentration gradient；And
It is substrate preparation into described that the series of oxidation trimethylamine working solution is respectively adopted into the human serum albumins aqueous solution
Series of oxidation trimethylamine titer.
In one of the embodiments, the concentration range of the series of oxidation trimethylamine working solution is 40~10000ng/
mL。
In one of the embodiments, in addition to according to the ratio that step (2) obtains and the TMAO mark
Relation between the TMAO concentration of quasi- liquid draws standard regression curve and carries out self-correcting to the standard regression curve
Step：
Each ratio that step (2) is obtained substitutes into the equation of the standard regression curve, obtains each TMAO
The concentration determination value of TMAO in titer；And
Judge whether each TMAO titer is calibration standard liquid, and according to each institute of the calibration standard liquid
Ratio and its concentration are stated, obtains the standard regression curve after self-correcting, wherein the quantity of the calibration standard liquid is not less than 6
It is individual；
Wherein, it is described to judge whether each TMAO titer is that calibration standard liquid specifically includes：If the serial oxygen
Changing the concentration determination value of the TMAO titer of least concentration and the ratio of the least concentration in trimethylamine titer is
80%~120%, then the TMAO titer of the least concentration is calibration standard liquid；If the or oxidation except least concentration
The ratio of the concentration determination value of TMAO and its concentration in other TMAO titers outside trimethylamine titer
For 85%~115%, then the TMAO titer is calibration standard liquid；
Correspondingly, step (3) obtains the test plasma sample according to the equation of the standard regression curve after self-correcting
The concentration of middle TMAO.
In one of the embodiments, the series of oxidation trimethylamine titer is also included before the step (2)
System suitability testing procedure：
Into the series of oxidation trimethylamine titer, the TMAO titer of least concentration adds internal standard compound and sunk
Shallow lake agent centrifuges to obtain supernatant, parallel to carry out multiple liquid chromatogram and mass spectral analysis, respectively obtains the oxygen of multiple least concentrations
Change the ratio of the chromatographic peak area of the TMAO of trimethylamine titer and the chromatographic peak area of the internal standard compound；
If the chromatographic peak area of the TMAO of the TMAO titer of multiple least concentrations with it is described interior
The coefficient of variation for marking the ratio of the chromatographic peak area of thing is less than preset value, then the series of oxidation front three in the step (1)
The concentration range of amine titer can be suitably used for the liquid chromatogram and the system of mass spectral analysis.
In one of the embodiments, the mobile phase that chromatography uses in the liquid chromatogram and mass spectral analysis is acetic acid
The acetonitrile solution of aqueous ammonium and formic acid, the chromatographic column are C18 chromatographic columns, and the flow velocity of the mobile phase is 0.2~0.8mL/
Min, the column temperature of the chromatographic column is 25~35 DEG C；The condition of mass spectral analysis is using electricity in the liquid chromatogram and mass spectral analysis
Electrospray ionization comes from cation MRM mode detections.
Brief description of the drawings
Fig. 1 is the standard regression curve for the TMAO titer that embodiment 1 obtains；
Fig. 2 is the chromatogram of the chromatogram of TMAO and the deuterated TMAO of internal standard compound in test plasma sample；
Fig. 3 is one of TMAO that the system suitability testing procedure of series of oxidation trimethylamine titer obtains
The chromatogram of the chromatogram of TMAO and internal standard compound in titer；
Fig. 4 is the chromatogram of TMAO and the deuterated oxidation front three of internal standard compound in blank human serum albumins aqueous solution E
The chromatogram of amine；
Fig. 5 is the chromatogram of TMAO and the deuterated oxidation front three of internal standard compound in blank human serum albumins aqueous solution F
The chromatogram of amine.
Embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In accompanying drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The implication that technical staff is generally understood that is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
The TMAO immue quantitative detection reagent box (not shown) of an embodiment of the present invention, including series of oxidation trimethylamine
Titer, internal standard compound and precipitating reagent.The solute of series of oxidation trimethylamine titer is TMAO, and solvent is human seralbumin egg
White water solution.
In one of the embodiments, the concentration range of series of oxidation trimethylamine titer is 2~500ng/mL.
In one of the embodiments, internal standard compound is deuterated TMAO, and precipitating reagent is acetonitrile.
In one of the embodiments, the quality of human serum albumins and the volume ratio of water in the human serum albumins aqueous solution
For (3~10) Kg:100L.
Above-mentioned TMAO immue quantitative detection reagent box, series of oxidation trimethylamine titer are water-soluble using human serum albumins
Liquid is matrix, so avoid using water, bovine serum albumin white liquor or phosphate buffer etc. and human plasma into phase-splitting difference compared with
Remote blank bio-matrix causes the obtained insecure problem of testing result, and then is advantageous to improve the reliable of testing result
Property.And using the internal standard method of internal standard compound, precipitated by precipitating reagent, be advantageous to further improve the reliability of testing result.
The TMAO quantitative detecting method of an embodiment of the present invention, examination is quantitatively detected using above-mentioned TMAO
Agent box, comprise the following steps (1)~(3).
Step (1)：TMAO is added in the human serum albumins aqueous solution and prepares the serial oxygen with concentration gradient
Change trimethylamine titer.
The human serum albumins aqueous solution is used as matrix, so avoids delaying using water, bovine serum albumin white liquor or phosphate
The blank bio-matrix into phase-splitting difference farther out of fliud flushing etc. and human plasma causes the obtained insecure problem of testing result, enters
And be advantageous to improve the reliability of testing result.
Preferably, the quality of human serum albumins and the volume ratio of water are (3~10) Kg in the human serum albumins aqueous solution:
100L.The content of human serum albumins is also close to be close to human plasma so in the control human serum albumins aqueous solution
Component content, further improve testing result reliability.
Preferably, the quantity of series of oxidation trimethylamine titer is no less than 6.It is highly preferred that series of oxidation trimethylamine mark
The quantity of quasi- liquid is 7~10.
In one of the embodiments, the human serum albumins aqueous solution is mixed with water by human serum albumins crystal and produced.
Further, 150~500mg human serum albumins crystal addition 5mL water is mixed and produced.Further, human seralbumin egg
There is provided in vain by Solarbio, lot number is：730A0521, purity are 96%~99%.Further, human serum albumins is water-soluble
Liquid is placed in -20 DEG C of preservations.
In one of the embodiments, step (1) comprises the following steps：TMAO is dissolved in into methanol aqueous solution to prepare
Series of oxidation trimethylamine working solution with concentration gradient；Human serum albumins is respectively adopted in series of oxidation trimethylamine working solution
The aqueous solution is substrate preparation into series of oxidation trimethylamine titer.
The series of oxidation trimethylamine working solution with concentration gradient is first prepared, then uses the human serum albumins aqueous solution as base
Matter is configured to series of oxidation trimethylamine titer, not only contributes to the series of oxidation trimethylamine working solution stablized, but also
Be advantageous to configuration and obtain that concentration is relatively low and accurate series of oxidation trimethylamine titer.
Further, the percent by volume of methanol is 30~60% in methanol aqueous solution.Preferably, first in methanol aqueous solution
The percent by volume of alcohol is 50%.
Further, the concentration range of series of oxidation trimethylamine working solution is 40~10000ng/mL.Further, it is
The concentration of row TMAO working solution is followed successively by 40,100,200,400,1000,2000,4000 and 10000ng/ from low to high
mL。
Preferably, TMAO is dissolved in methanol aqueous solution and prepares the series of oxidation trimethylamine work with concentration gradient
Liquid, diluted successively using high concentration, the mode for obtaining low concentration configures.The uniformity of solution is not only so ensure that, and is subtracted
The small multiple for needing to dilute in the TMAO working solution of subsequent configuration low concentration, is advantageous to improve series of oxidation front three
The accuracy of the concentration of amine working solution.
Specifically in one embodiment, TMAO is dissolved in methanol aqueous solution preparation 1mg/mL TMAO first
Stoste, dilute 100 times of TMAO working solutions for obtaining 10000ng/mL.10000ng/mL TMAO working solution is dilute
Release the 2.5 times of TMAO for obtaining 4000ng/mL working solutions.The rest may be inferred.
Preferably, series of oxidation trimethylamine working solution is configured to times diluted during series of oxidation trimethylamine titer respectively
Number is identical.Further, series of oxidation trimethylamine working solution is configured to what is diluted during series of oxidation trimethylamine titer respectively
Multiple is 20 times.
Further, the concentration range of series of oxidation trimethylamine titer is 2~500ng/mL.Further, it is serial
The concentration of TMAO titer is followed successively by 2,5,10,20,50,100,200 and 500ng/mL from low to high.So using this
The lower limit of the concentration of the detectable TMAO of method can reach 2ng/mL, and higher limit reaches 500ng/mL, therefore should
The detection range of method is wide, practical.
Step (2)：Series of oxidation trimethylamine titer is added into internal standard compound and precipitating reagent centrifuges to obtain supernatant, is carried out respectively
Liquid chromatogram and mass spectral analysis, obtain the chromatographic peak area of TMAO and the color of internal standard compound in each TMAO titer
The ratio of spectral peak area, and obtain the relation between ratio and the TMAO concentration of TMAO titer.
In one of the embodiments, in addition to according to step (2) ratio and the oxygen of TMAO titer obtained
Change the step of relation between trimethylamine concentration draws standard regression curve.The equation of standard regression curve is：Y=aX+b.
Wherein a is standard regression slope of a curve, and b is the intercept of standard regression curve, and Y is in TMAO titer
The ratio of the chromatographic peak area of TMAO and the chromatographic peak area of internal standard compound, X are to aoxidize three in TMAO titer
The concentration of methylamine, unit ng/mL.
Internal standard method is so used, and using the chromatographic peak area and internal standard compound of TMAO in TMAO titer
Chromatographic peak area ratio for Y value carry out linear regression, obtain standard regression curve, be advantageous to further improve testing result
Reliability.
Specifically, centrifugation step centrifuges after using vortex in step (2).
Preferably, the coefficient correlation of the equation of standard regression curve is not less than 0.99.
In one of the embodiments, in addition to standard regression curve the step of carrying out self-correcting：Step (2) is obtained
Each ratio substitutes into the equation of standard regression curve, obtains the test value of TMAO in each TMAO titer；Judge
Whether each TMAO titer is calibration standard liquid, and according to each ratio and its concentration of calibration standard liquid, obtains self-correcting
Standard regression curve afterwards, the wherein quantity of calibration standard liquid are not less than 6.
Wherein, judge whether each TMAO titer is that calibration standard liquid specifically includes：If series of oxidation trimethylamine
The ratio of the concentration determination value of the TMAO titer of least concentration and least concentration is 80%~120% in titer,
Then the TMAO titer of least concentration is calibration standard liquid；If or in addition to the TMAO titer of least concentration
Other TMAO titers in the ratio of concentration determination value and its concentration of TMAO be 85%~115%, then
The TMAO titer is calibration standard liquid.
It is appreciated that do not meet the TMAO titer of calibration standard liquid, then for can not calibration standard liquid；And then will
This can not calibration standard liquid reject, front three is aoxidized with corresponding TMAO titer according to each ratio of calibration standard liquid
The concentration of amine, retrieve the standard regression curve after self-correcting.Correspondingly, step (3) is according to the standard regression curve after self-correcting
Equation obtain the concentration of TMAO in test plasma sample.Can not school if the series of oxidation trimethylamine titer is not present
Positive titer, then the standard regression curve after self-correcting is primary standard regression curve.
In one of the embodiments, can not be after calibration standard liquid if rejecting, the quantity of calibration standard liquid is less than 6, then
Need to re-start step (1), to ensure the uniformity of series of oxidation trimethylamine titer.
In one of the embodiments, also the system including series of oxidation trimethylamine titer is applicable before step (2)
Property testing procedure：
Into series of oxidation trimethylamine titer, the TMAO titer of least concentration adds internal standard compound and precipitating reagent
Supernatant is centrifuged to obtain, it is parallel to carry out multiple liquid chromatogram and mass spectral analysis, respectively obtain the TMAO of multiple least concentrations
The ratio of the chromatographic peak area of the TMAO of titer and the chromatographic peak area of internal standard compound；
If the chromatographic peak area of TMAO and the color of internal standard compound of the TMAO titer of multiple least concentrations
The coefficient of variation of the ratio of spectral peak area is less than preset value, then the concentration model of the series of oxidation trimethylamine titer in step (1)
Enclose the system that can be suitably used for liquid chromatogram and mass spectral analysis.
By carrying out multiple liquid phase color to the TMAO titer of least concentration in series of oxidation trimethylamine titer
Spectrum and mass spectral analysis, judge the suitable of the TMAO titer of the least concentration and the liquid chromatogram and the system of mass spectral analysis
The property used.If the TMAO titer of the least concentration is good with the applicability of the liquid chromatogram and the system of mass spectral analysis,
Then illustrate that the series of oxidation trimethylamine titer in step (1) is applied to liquid chromatogram and the system of mass spectral analysis.This be because
For the coefficient of variation TMAO titer that is bigger, therefore passing through least concentration of the lower TMAO titer of concentration
The coefficient of variation be that can determine that the applicability of the series of oxidation trimethylamine titer and the liquid chromatogram and the system of mass spectral analysis.
Further, the calculation formula of the coefficient of variation is：The coefficient of variation=(standard deviation/average value) × 100%.
Further, the preset value is 10%.
Preferably, the parallel liquid chromatogram and the number of mass spectral analysis of carrying out is 6 times, the obtained TMAO titer
The quantity of the ratio of the chromatographic peak area of middle TMAO and the chromatographic peak area of internal standard compound is 6.
In one of the embodiments, when the concentration range of series of oxidation trimethylamine titer determines, by step (1) and
Step (2) is parallel to be carried out repeatedly, respectively obtaining multiple standard regression curves.Further, step (1) it is parallel with step (2) enter
Capable number is three times, to respectively obtain three standard regression curves.
Step (3)：Test plasma sample is added into internal standard compound and precipitating reagent centrifuges to obtain supernatant, carries out liquid chromatogram and matter
Spectrum analysis, obtains the ratio of the chromatographic peak area of the chromatographic peak area of TMAO and internal standard compound in test plasma sample, and
Relation between the ratio and the TMAO concentration of the TMAO titer that are obtained according to step (2), is obtained
The concentration of TMAO into test plasma sample.
Supernatant is obtained after internal standard compound and precipitating reagent centrifugation pretreatment are so added to plasma sample to be measured, passes through a liquid phase
Chromatogram and mass spectral analysis and step (2) are obtained between the TMAO concentration of the ratio and the TMAO titer
Relation, can quickly obtain the concentration of the TMAO of test plasma sample, avoid and answered using long column or liquid-phase condition
The miscellaneous sample analysis time for causing method to detect length, it is impossible to the problem of meeting the requirement of Big Clinical Samples amount quick detection, be blood
Quantifying for TMAO concentration provides a kind of fast and accurately high-sensitivity detecting method in slurry sample.
The degree of accuracy and the precision of Method validation testing result have been carried out in the form of recovery of standard addition, has been illustrated above-mentioned
TMAO quantitative detecting method improves the degree of accuracy and the precision height of testing result.And step (1) and step (2) step
Rapid simple, time-consuming short, sample introduction analysis time of each test plasma sample of step (3) is 1.5min, each test plasma sample
The total process of test can be completed in 30 minutes, substantially reduce analysis detection time, and then meet the quick of high-volume sample
The requirement of detection.
In addition, by Method validation, using above-mentioned TMAO quantitative detecting method, the content of TMAO
Monitoring lower-cut reaches 2ng/mL, and the reliability of testing result and the degree of accuracy are high, disclosure satisfy that the inspection of low concentration clinical blood sample
Surveying needs.
Further, in step (2) and step (3), internal standard compound is deuterated TMAO, D9-TMAO.Preferably,
Internal standard compound is added in the form of its methanol aqueous solution.Specifically, the methanol aqueous solution in the methanol aqueous solution of the internal standard compound, its first
The percent by volume of alcohol is 30~60%.Preferably, the percent by volume of methanol is 50% in methanol aqueous solution.It is specifically real one
Apply in example, the concentration of the internal standard compound in the methanol aqueous solution of internal standard compound is 21ng/mL.
In one of the embodiments, precipitating reagent is acetonitrile.Preferably, the volume of precipitating reagent and TMAO titer
Volume ratio, or the volume ratio of the volume of precipitating reagent and test plasma sample is 2~5：1.It is highly preferred that the volume of precipitating reagent
With the volume ratio of TMAO titer, or the volume ratio of the volume of precipitating reagent and test plasma sample is 2.5：1.
Specifically in one embodiment, TMAO titer and each μ L of sample introduction 3 of test plasma sample.
Preferably, precipitating reagent is added using vibration and centrifugal treating, obtains supernatant.It is highly preferred that the time of vibration is
10~20s, the speed of centrifugation is 8000~13000rpm.Specifically, centrifugation step centrifuges after using vortex in step (3).
In one of the embodiments, the mobile phase that chromatography uses in liquid chromatogram and mass spectral analysis is ammonium acetate water
The acetonitrile solution of solution and formic acid, chromatographic column are C18 chromatographic columns, and the flow velocity of mobile phase is 0.2~0.8mL/min, chromatographic column
Column temperature is 25~35 DEG C.In one of the embodiments, the condition of mass spectral analysis is using electricity in liquid chromatogram and mass spectral analysis
Electrospray ionization comes from cation MRM mode detections.
Preferably, the volume ratio of the acetonitrile solution of ammonium acetate solution and formic acid is 20%~40%:80~60%.It is more excellent
Selection of land, the concentration of ammonium acetate solution is 10mmol/L, and the percent by volume of the acetonitrile solution of formic acid is 0.1%.
It is specific embodiment below.
Embodiment 1
1) instrument and equipment
Waters TQ-D mass spectrometer systems；Waters UPLC I-Class liquid phase systems；Waters UNIFI softwares；
Thermo Fisher Thermo ST16R types high speed freezing centrifuge (match is silent to fly, the U.S.)；XW-80A turbine mixers (Ningbo
Xin Zhi biotech inc)；Pipettor (Gilson).
2) reagent and material
TMAO：Tokyo HuaCheng Industry Co., Ltd, purity 99.5%, lot number：3EVJG-MC；Deuterated oxidation front three
Amine：Toronto Research Chemicals, purity 98%, lot number：3-LXM-155-1；Ammonium acetate：Pure, traditional Chinese medicines are analyzed, batch
Number：20160310；Acetonitrile：Gradient grade for liquid chromatography, Merck；Formic acid：LC levels,
ACS；Human serum albumins：Purity 96%-99%, Solarbio, lot number：730A0521；
3) liquid chromatogram and mass spectral analysis condition
Chromatographic condition
Chromatographic column：ACQUITYBEH C18(2.1 × 50mm, 1.7 μm)；Mobile phase：10mM ammonium acetate solutions:
Acetonitrile solution=80 of the formic acid of percent by volume 0.1%:20(v/v)；Flow velocity 0.2mL/min；30 DEG C of column temperature；Sample introduction room temperature：
10℃；The μ L of sample size 3；Wash pin liquid：80% methanol-water.
Mass Spectrometry Conditions
Using electric spray ion source (ESI), cation MRM mode detections；TMAO：Monitor ion m/z 75.99
→m/z 58.6；Taper hole voltage：18V, impact energy：12eV；TMAO-D9：Monitor 85.1 → m/z of ion m/z 68, cone
Hole voltage：18V, impact energy：13eV；Capillary voltage：0.5kV；Taper hole air-flow：60L/h；Go solvent stream：1000L/h；From
Source temperature：150℃；Remove solvent temperature：500℃.
4) solution is prepared
Configure TMAO storing solution I：Precision weighs TMAO crystal 10.05mg, through purity factor correction meter
TMAO weight is obtained after calculation is：10mg.10mL volumetric flasks are settled to the methanol-water that percent by volume is 50%, are obtained
1mg/mL TMAO storing solution, it is placed in -20 DEG C of refrigerators and preserves.
Configure TMAO storing solution II：Precision weighs TMAO crystal 10.05mg, through purity factor correction meter
TMAO weight is obtained after calculation is：10mg.1L volumetric flasks are settled to 50% methanol-water, it follows that standard concentration is
0.01mg/mL, it is placed in -20 DEG C of refrigerators and preserves.
Configure series of oxidation trimethylamine working solution：Dilute to obtain using 1mg/mL TMAO storing solution I
10000ng/mL working solution 8,4000ng/mL working solution 7 is obtained using the dilution of working solution 8, the rest may be inferred, as shown in table 1,
Wherein 50% methanol-water is the methanol aqueous solution that percent by volume is 50%.
Table 1
Configure deuterated TMAO storing solution：Precision weighs TMAO-D9Reference substance powder 1mg, through purity school
Positive divisor obtains TMAO-D after calculating9Standard items weight：1.06mg, standard items are settled to 25mL capacity with 50% methanol-water
Bottle, obtains 42 μ g/mL deuterated TMAO storing solution, is placed in -20 DEG C of preservations.
Configure the mixed liquor of deuterated TMAO storing solution and acetonitrile：Pipette 42 μ g/mL deuterated TMAO storage
The standby μ L of liquid 80, add 160mL acetonitriles and mix, be placed in solvent bottle, after ultrasonic 5min.
Configure 3~10% (quality of human serum albumins and the volume ratio of water, Kg/L) human serum albumins aqueous solution：Make
150~500mg human serum albumins crystal is weighed with one thousandth balance add 5mL ultra-pure waters and mix and produce, be placed in -20 DEG C of guarantors
Deposit.
Mobile phase is prepared：The configuration of aqueous phase：Precision measures 100mL deionization ultra-pure waters, adds 0.385g ammonium acetates, mixes
400mL ultra-pure waters are added to mix ultrasonic 5min again with 0.22 μm of membrane filtration, filtrate afterwards.The configuration of organic phase：Precision measures
200mL acetonitriles, add 200 μ L formic acid, mixing.
Prepare series of oxidation trimethylamine titer：Precision pipettes 1~8 each 5 μ L of series of oxidation trimethylamine working solution, adds 95
The μ L 10% human serum albumins aqueous solution, mix, obtain concentration and be followed successively by 2,5,10,20,50,100,200 and 500ng/mL
Series of oxidation trimethylamine titer.
5) TMAO titer or the sample introduction pre-treatment of test plasma sample and sample introduction analysis
Sample introduction pre-treatment：It is accurate respectively to pipette the series that concentration is followed successively by 2,5,10,20,50,100,200 and 500ng/mL
TMAO titer or the μ L of test plasma sample 100, add the mixing of the deuterated TMAO storing solutions of 250 μ L and acetonitrile
Liquid, 10s is vibrated, 13000rpm centrifugation 5min, takes the μ L of supernatant 100, respectively the μ L of sample introduction 3 to liquid chromatogram and mass spectrometry system
Analyzed.
Series of oxidation trimethylamine titer sample introduction is analyzed：Obtain the chromatogram of TMAO in each TMAO titer
The ratio of peak area and the chromatographic peak area of the internal standard compound；And obtain the oxygen of the ratio and the TMAO titer
Change the relation between trimethylamine concentration, and draw standard regression curve, as shown in Figure 1.
Wherein, the equation of standard regression curve is：Y=aX+b；A is standard regression slope of a curve, and b is that standard regression is bent
The intercept of line, Y are the chromatographic peak of the chromatographic peak area of TMAO and the internal standard compound in the TMAO titer
The ratio of area, X be the TMAO titer in TMAO concentration, unit ng/mL.Specifically, Fig. 1 is obtained
The equation of the standard regression curve arrived is Y=0.00569X+0.00376, coefficient R2=0.999471.
Test plasma sample sample introduction is analyzed：It is deuterated to obtain the chromatographic peak of TMAO and internal standard compound in test plasma sample
The chromatographic peak of TMAO is respectively as shown in A in Fig. 2 and B, and therefrom obtain the color of TMAO in test plasma sample
The ratio of spectral peak area and the chromatographic peak area of the internal standard compound is 0.495.
According to the equation Y=0.00569X+0.00376 of standard regression curve, obtain aoxidizing front three in test plasma sample
The concentration of amine is 86.4ng/mL.
6) the self-correcting step of the equation of standard regression curve：
Each ratio is substituted into the equation of standard regression curve, obtains the survey of TMAO in each TMAO titer
Examination value.Compare in 2ng/mL TMAO titer the test value of TMAO and 2ng/mL ratio 80%~
Within 120%, compare in other TMAO titers in addition to 2ng/mL the test value of TMAO with it is corresponding
The ratio of the concentration of TMAO is within 85%~115% in TMAO titer, then the series of oxidation trimethylamine
Titer is calibration standard liquid, without can not calibration standard liquid, therefore the standard regression curve after self-correcting is original mark
Quasi- regression curve, illustrate that the degree of accuracy of original standard regression curve is very high, TMAO in obtained test plasma sample
The concentration degree of accuracy it is high.
7) the system suitability testing procedure of series of oxidation trimethylamine titer：
Sample introduction pre-treatment：Precision pipettes the μ L of TMAO titer 100 that concentration is 2ng/mL, and it is deuterated to add 250 μ L
The mixed liquor of TMAO storing solution and acetonitrile, 10s is vibrated, 13000rpm centrifugation 5min, the μ L of supernatant 100 is taken, enters respectively
The μ L of sample 3 are analyzed to liquid chromatogram and mass spectrometry system.Repeat sample introduction 6 times, respectively obtain 6 TMAO standards
The ratio of the chromatographic peak area of the chromatographic peak area of TMAO and internal standard compound in liquid.And obtain the standard deviation of this 6 ratios
Difference and average value, and then the ratio i.e. coefficient of variation for obtaining standard deviation and average value is less than 10%.Illustrate that detecting instrument makes
Showed when detecting TMAO with this method very stable.
Wherein, the chromatographic peak of TMAO and the chromatographic peak of internal standard compound in the TMAO titer, respectively
As shown in Fig. 3 C and D, the retention time that can therefrom learn TMAO and the deuterated TMAO of internal standard compound is respectively
0.537 and 0.534, the ratio of the chromatographic peak area of TMAO and the chromatographic peak area of internal standard compound is 31204/2057343
=0.015167.
8) method validation is tested
Matrix interference is tested：Separately take 2 part of 10% human serum albumins aqueous solution, every part of 100 μ L.1 part with acetonitrile precipitation, note
For E.The mixing liquid precipitate of the deuterated TMAO storing solution of another 1 part of use and acetonitrile, is designated as F.Vibrate 10s, 13000rpm centrifugations
5min, the μ L of supernatant 100 are taken, the μ L of sample introduction 3 are analyzed to liquid chromatogram and mass spectrometry system respectively.E and F two is recorded respectively
The chromatogram of part blank human serum albumins aqueous solution, for evaluating the interference feelings of the human serum albumins aqueous solution of blank 10%
Condition；Obtained chromatogram difference is as shown in Figures 4 and 5.Fig. 4 E1 and E2 corresponds to TMAO and deuterated oxidation front three respectively
The chromatographic peak of amine, Fig. 5 F1 and F2 correspond to the chromatographic peak of TMAO and deuterated TMAO respectively.
It can be seen that from Fig. 4 E1 and E2 in the human serum albumins aqueous solution without TMAO and deuterated oxidation three
The peak value of the retention time of methylamine.It can be seen that there was only deuterated oxidation in the human serum albumins aqueous solution from Fig. 5 middle F1 and F2
The peak value of the retention time of trimethylamine, without the peak value of the retention time of TMAO.Therefore explanation two parts of blank of E and F
Interference is not present in test of the human serum albumins aqueous solution to TMAO.
Residue test：Accurate TMAO titer, the 2ng/mL TMAO mark for pipetting 500ng/mL respectively
The quasi- μ L of liquid 100, the mixed liquor of the deuterated TMAO storing solutions of 250 μ L and acetonitrile is added, vibrate 10s, 13000rpm centrifugations
5min, take the μ L of supernatant 100.1 part of 10% μ L of human serum albumins aqueous solution blank sample 150 is handled simultaneously, adds 250 μ L second
Nitrile, 10s is vibrated, 13000rpm centrifugation 5min, takes the μ L of supernatant 100.By upper limit of quantification (500ng/mL), dummy, quantify
The order sample introduction of lower limit (2ng/mL), investigate the residual condition of instrument and chromatographic column.
Residue test result illustrates that the residual concentration in blank sample is less than the 20% of lower limit of quantitation, and no more than deuterated
The 5% of TMAO concentration.Meet residual acceptable standard：Residual after upper limit of quantification sample in blank sample should not
More than the 20% of lower limit of quantitation, and it is no more than interior target 5%.
Precision is tested with accuracy validation：
Diluted successively using TMAO storing solution II respectively obtain 8000ng/mL high concentration TMAO it is molten
Liquid, 800ng/mL middle concentration TMAO solution and 80ng/mL high concentration oxidation trimethylamine solution, as shown in table 2, its
In 50% methanol-water be methanol aqueous solution that percent by volume is 50%.
Table 2
The TMAO solution of above-mentioned high, medium and low concentration is respectively taken into 5 μ L, adds 95 μ L 10% human serum albumins
The aqueous solution, mix, obtain the TMAO solution that concentration is followed successively by 400ng/mL, 40ng/mL and 4ng/mL.
According to the equation Y=0.00569X+0.00376 of standard regression curve, coefficient R2=0.999471 obtains oxygen
Change plasma sample of the trimethylamine concentration in 2~4ng/mL.The blood sample sample is divided into three groups, is separately added into 400ng/mL, 40ng/
ML and 4ng/mL TMAO solution obtains sample after mark-on as mark-on sample.Every group parallel 6 parts, 100 μ L are taken respectively,
The mixed liquor of the deuterated TMAO storing solutions of 250 μ L and acetonitrile is added, 10s is vibrated, 13000rpm centrifugation 5min, takes supernatant
100 μ L, respectively the μ L of sample introduction 3 analyzed to liquid chromatogram and mass spectrometry system.
According to the equation Y=0.00569X+0.00376 of standard regression curve, the test concentrations of sample after mark-on are obtained.Root
Every group of the coefficient of variation and average value are calculated according to the sign concentration of sample after the test concentrations and mark-on of sample after mark-on, wherein adding
The sign concentration of sample is the concentration of plasma sample and the concentration sum of mark-on sample after mark.Wherein the coefficient of variation is standard deviation
With the ratio of average value, its big I characterization accuracy.Average value can characterize the degree of accuracy.Result of the test shows, the variation of each group
For coefficient not less than 15%, the average value of each group in the range of (100% ± 15%) of the sign concentration of sample, meets mark after mark-on
Standard, illustrate the test plasma obtained using the equation Y=0.00569X+0.00376 and the above method of above-mentioned standard regression curve
The accuracy and precision of the test concentrations of sample is very high.
8) dilution process is tested
10 times of dilution：
Precision pipettes 100 μ L human plasma samples, parallel 6 parts.Precision pipettes adds 10% with a μ L of human plasma sample 10
The μ L of the human serum albumins aqueous solution 90, mix, must dilute 10 times of human plasma sample, parallel 6 parts.By undiluted human plasma sample
Sheet and the human plasma sample of 10 times of dilution are separately added into the mixed liquor of the deuterated TMAO storing solutions of 250 μ L and acetonitrile, vibration
10s, 13000rpm centrifuge 5min, take the μ L of supernatant 100, the μ L of sample introduction 3 are divided to liquid chromatogram and mass spectrometry system respectively
Analysis.Respectively obtain undiluted human plasma sample and dilute 10 times human plasma sample TMAO chromatographic peak area with
The ratio of the chromatographic peak area of deuterated TMAO.
According to the equation Y=0.00569X+0.00376 of above-mentioned standard regression curve, undiluted human plasma is calculated
The test concentrations of sample and the human plasma sample of 10 times of dilution, the test concentrations for further obtaining undiluted human plasma sample are put down
The test concentrations average value of average and the human plasma sample of 10 times of dilution.
The test concentrations average value for the human plasma sample for diluting 10 times is multiplied by extension rate and undiluted human plasma sample
This test concentrations average value compares, and result of the test shows, the test concentrations average value of the human plasma sample of 10 times of dilution is multiplied by
Extension rate meets dilution in the range of (100% ± 15%) of the test concentrations average value of undiluted human plasma sample
10 times receive scope.
20 times of dilution：
Precision pipettes 100 μ L human plasma samples, parallel 6 parts.Precision pipettes adds 10% with a μ L of human plasma sample 10
The μ L of the human serum albumins aqueous solution 190, mix, must dilute 20 times of human plasma sample, parallel 6 parts.By undiluted human plasma
Sample and the human plasma sample of 10 times of dilution are separately added into the mixed liquor of the deuterated TMAO storing solutions of 250 μ L and acetonitrile, shake
10s is swung, 13000rpm centrifugation 5min, takes the μ L of supernatant 100, the μ L of sample introduction 3 to liquid chromatogram and mass spectrometry system are carried out respectively
Analysis.Respectively obtain the chromatographic peak area of the TMAO of undiluted human plasma sample and the human plasma sample of 20 times of dilution
With the ratio of the chromatographic peak area of deuterated TMAO.
According to the equation Y=0.00569X+0.00376 of above-mentioned standard regression curve, undiluted human plasma is calculated
The test concentrations of sample and the human plasma sample of 20 times of dilution, the test concentrations for further obtaining undiluted human plasma sample are put down
The test concentrations average value of average and the human plasma sample of 20 times of dilution.
The test concentrations average value for the human plasma sample for diluting 20 times is multiplied by extension rate and undiluted human plasma sample
This test concentrations average value compares, and result of the test shows, the test concentrations average value of the human plasma sample of 20 times of dilution is multiplied by
Extension rate meets dilution in the range of (100% ± 15%) of the test concentrations average value of undiluted human plasma sample
20 times receive scope.
Thus illustrating, the concentration of TMAO is diluted using human serum albumins liquid in test plasma sample, its
Concentration also has the reliability to be converted by extension rate.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of TMAO immue quantitative detection reagent box, it is characterised in that including series of oxidation trimethylamine titer, internal standard compound
And precipitating reagent；The solute of the series of oxidation trimethylamine titer is TMAO, the solvent behaviour seralbumin aqueous solution.
2. TMAO immue quantitative detection reagent box as claimed in claim 1, it is characterised in that the series of oxidation trimethylamine
The concentration range of titer is 2~500ng/mL.
3. TMAO immue quantitative detection reagent box as claimed in claim 1, it is characterised in that the internal standard compound is deuterated oxygen
Change trimethylamine, the precipitating reagent is acetonitrile.
4. the TMAO immue quantitative detection reagent box as described in any one of claims 1 to 3, it is characterised in that people's blood
The quality of human serum albumins and the volume ratio of water are (3~10) Kg in pure protein solution:100L.
5. a kind of TMAO quantitative detecting method, is quantified using the TMAO as described in any one of Claims 1 to 4
Detection kit, it is characterised in that comprise the following steps：
Step (1), series of oxidation three of the TMAO preparation with concentration gradient is added in the human serum albumins aqueous solution
Methylamine titer；
Step (2), the series of oxidation trimethylamine titer is added into internal standard compound and precipitating reagent centrifuge to obtain supernatant, carry out respectively
Liquid chromatogram and mass spectral analysis, obtain the chromatographic peak area of TMAO and the internal standard compound in each TMAO titer
Chromatographic peak area ratio, and obtain between the TMAO concentration of the ratio and the TMAO titer
Relation；
Step (3), the test plasma sample addition internal standard compound and the precipitating reagent are centrifuged to obtain to supernatant, progress liquid chromatogram
And mass spectral analysis, obtain the chromatographic peak face of the chromatographic peak area of TMAO and the internal standard compound in the test plasma sample
Long-pending ratio, and the TMAO concentration of the ratio obtained according to step (2) and the TMAO titer it
Between relation, obtain the concentration of TMAO in the test plasma sample.
6. TMAO quantitative detecting method as claimed in claim 5, it is characterised in that the step (1) includes following
Step：
TMAO is dissolved in methanol aqueous solution and prepares the series of oxidation trimethylamine working solution with concentration gradient；And
It is substrate preparation into the series that the series of oxidation trimethylamine working solution is respectively adopted into the human serum albumins aqueous solution
TMAO titer.
7. TMAO quantitative detecting method as claimed in claim 6, it is characterised in that the series of oxidation trimethylamine work
The concentration range for making liquid is 40~10000ng/mL.
8. TMAO quantitative detecting method as claimed in claim 5, it is characterised in that also include being obtained according to step (2)
Relation between the ratio and the TMAO concentration of the TMAO titer that arrive draws standard regression curve
And the step of self-correcting is carried out to the standard regression curve：
Each ratio that step (2) is obtained substitutes into the equation of the standard regression curve, obtains each TMAO standard
The concentration determination value of TMAO in liquid；And
Judge whether each TMAO titer is calibration standard liquid, and according to each ratio of the calibration standard liquid
Value and its concentration, obtain the standard regression curve after self-correcting, wherein the quantity of the calibration standard liquid is not less than 6；
Wherein, it is described to judge whether each TMAO titer is that calibration standard liquid specifically includes：If the series of oxidation three
The ratio of the concentration determination value of the TMAO titer of least concentration and the least concentration is 80% in methylamine titer
~120%, then the TMAO titer of the least concentration is calibration standard liquid；If the or oxidation front three except least concentration
The ratio of the concentration determination value and its concentration of TMAO is in other TMAO titers outside amine titer
85%~115%, then the TMAO titer is calibration standard liquid；
Correspondingly, step (3) obtains oxygen in the test plasma sample according to the equation of the standard regression curve after self-correcting
Change the concentration of trimethylamine.
9. TMAO quantitative detecting method as claimed in claim 5, it is characterised in that before the step (2) also
Include the system suitability testing procedure of the series of oxidation trimethylamine titer：
Into the series of oxidation trimethylamine titer, the TMAO titer of least concentration adds internal standard compound and precipitating reagent
Supernatant is centrifuged to obtain, it is parallel to carry out multiple liquid chromatogram and mass spectral analysis, respectively obtain the oxidation three of multiple least concentrations
The ratio of the chromatographic peak area of the TMAO of methylamine titer and the chromatographic peak area of the internal standard compound；
If the chromatographic peak area of the TMAO of the TMAO titer of multiple least concentrations and the internal standard compound
The coefficient of variation of ratio of chromatographic peak area be less than preset value, then the series of oxidation trimethylamine mark in the step (1)
The concentration range of quasi- liquid can be suitably used for the liquid chromatogram and the system of mass spectral analysis.
10. the TMAO quantitative detecting method as described in any one of claim 5~9, it is characterised in that the liquid phase color
Compose the mobile phase used with chromatography in mass spectral analysis is for ammonium acetate solution and the acetonitrile solution of formic acid, the chromatographic column
C18 chromatographic columns, the flow velocity of the mobile phase is 0.2~0.8mL/min, and the column temperature of the chromatographic column is 25~35 DEG C；The liquid
The condition of mass spectral analysis is in cation MRM mode detections using electric spray ion source in phase chromatogram and mass spectral analysis.
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| CN108507984A (en) * | 2018-02-28 | 2018-09-07 | 山东大学第二医院 | A kind of method and its application of enzyme process detection trimethylamine oxide TMAO |
| CN109163951A (en) * | 2018-11-05 | 2019-01-08 | 长沙都正医学检验有限责任公司 | A kind of preparation facilities of TMAO negative sample |
| CN109917042A (en) * | 2019-04-08 | 2019-06-21 | 常州市第一人民医院 | A kind of kit |
| CN110487929A (en) * | 2019-08-16 | 2019-11-22 | 长春中医药大学 | A method of utilizing trimethylamine oxide content in reaction assay human serum in ml headspace bottle |
| CN110554117A (en) * | 2019-10-09 | 2019-12-10 | 南方医科大学南方医院 | Application of trimethylamine oxide as stroke biomarker |
| CN111351885A (en) * | 2018-12-20 | 2020-06-30 | 长沙都正生物科技有限责任公司 | Biological negative sample for TMAO detection and preparation method and application thereof |
| CN111650285A (en) * | 2019-09-24 | 2020-09-11 | 上海透景诊断科技有限公司 | Detection method and detection kit for content of trimethylamine oxide in blood plasma |
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| CN111983112A (en) * | 2020-09-09 | 2020-11-24 | 南京诺禾致源生物科技有限公司 | Detection method of TMAO and related metabolites thereof in serum |
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| CN112601960A (en) * | 2018-08-13 | 2021-04-02 | 吴明贤 | Detection method for measuring in-vivo trimethylamine oxide productivity |
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| CN113376267A (en) * | 2021-05-11 | 2021-09-10 | 兰立生物科技(苏州)有限公司 | Method and kit capable of rapidly detecting TMAO biomarker |
| CN113686998A (en) * | 2021-09-28 | 2021-11-23 | 江苏晗睿达精准医学科技有限公司 | Method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101046479A (en) * | 2007-02-25 | 2007-10-03 | 清华大学 | Process of preparing human serum base matter containing no target protein |
| CN102539595A (en) * | 2010-12-09 | 2012-07-04 | 北京国立柏林医学科技发展有限公司 | Method for detecting content of isosuccinic acid in blood or urine |
| CN103197084A (en) * | 2013-03-28 | 2013-07-10 | 山东博科生物产业有限公司 | Stable glycated serum protein detection reagent and application thereof |
| CN105002264A (en) * | 2015-07-28 | 2015-10-28 | 中生北控生物科技股份有限公司 | Total bile acid measuring kit |
| CN106018852A (en) * | 2011-09-20 | 2016-10-12 | 生物辐射实验室股份有限公司 | Customized quality controls for analytical assays |
| CN106556656A (en) * | 2016-10-26 | 2017-04-05 | 南京医科大学 | A kind of blood plasma metabolism small molecule mark related to cholelithiasis and its application |
-
2017
- 2017-06-27 CN CN201710502265.5A patent/CN107340341A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101046479A (en) * | 2007-02-25 | 2007-10-03 | 清华大学 | Process of preparing human serum base matter containing no target protein |
| CN102539595A (en) * | 2010-12-09 | 2012-07-04 | 北京国立柏林医学科技发展有限公司 | Method for detecting content of isosuccinic acid in blood or urine |
| CN106018852A (en) * | 2011-09-20 | 2016-10-12 | 生物辐射实验室股份有限公司 | Customized quality controls for analytical assays |
| CN103197084A (en) * | 2013-03-28 | 2013-07-10 | 山东博科生物产业有限公司 | Stable glycated serum protein detection reagent and application thereof |
| CN105002264A (en) * | 2015-07-28 | 2015-10-28 | 中生北控生物科技股份有限公司 | Total bile acid measuring kit |
| CN106556656A (en) * | 2016-10-26 | 2017-04-05 | 南京医科大学 | A kind of blood plasma metabolism small molecule mark related to cholelithiasis and its application |
Non-Patent Citations (5)
| Title |
|---|
| DANIEL M. MUELLER ET AL.: "Plasma levels of trimethylamine-N-oxide are confounded by impaired kidney function and poor metabolic control", 《ATHEROSCLEROSIS》 * |
| HANANE KADAR ET AL.: "A multiplexed targeted assay for high-throughput quantitative analysis of serum methylamines by ultra performance liquid chromatography coupled to high resolution mass spectrometry", 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 * |
| ROBYN J. MACLELLAN ET AL.: "Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogish shark (Squalus acanthias)", 《J COMP PHYSIOL B》 * |
| 刘燕 等: "血清中氧化三甲胺的高效液相色谱串联质谱法测定研究", 《华南预防医学》 * |
| 张彦 等: "《保健食品代谢动力学》", 31 October 2016, 电子科技大学出版社 * |
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| CN110487929A (en) * | 2019-08-16 | 2019-11-22 | 长春中医药大学 | A method of utilizing trimethylamine oxide content in reaction assay human serum in ml headspace bottle |
| CN111650285A (en) * | 2019-09-24 | 2020-09-11 | 上海透景诊断科技有限公司 | Detection method and detection kit for content of trimethylamine oxide in blood plasma |
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| CN110554117A (en) * | 2019-10-09 | 2019-12-10 | 南方医科大学南方医院 | Application of trimethylamine oxide as stroke biomarker |
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| CN111983112A (en) * | 2020-09-09 | 2020-11-24 | 南京诺禾致源生物科技有限公司 | Detection method of TMAO and related metabolites thereof in serum |
| CN113176364A (en) * | 2021-04-19 | 2021-07-27 | 中国医学科学院北京协和医院 | Method for simultaneously detecting trimethylamine oxide and phenylacetylglutamine, detection kit and application thereof |
| CN113376267A (en) * | 2021-05-11 | 2021-09-10 | 兰立生物科技(苏州)有限公司 | Method and kit capable of rapidly detecting TMAO biomarker |
| CN113686998A (en) * | 2021-09-28 | 2021-11-23 | 江苏晗睿达精准医学科技有限公司 | Method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology |
| CN114371239A (en) * | 2022-03-22 | 2022-04-19 | 长沙都正医学检验有限责任公司 | Kit for determining trimethylamine oxide and preparation method and application thereof |
| CN114371239B (en) * | 2022-03-22 | 2022-07-15 | 长沙都正医学检验有限责任公司 | Kit for determining trimethylamine oxide and preparation method and application thereof |
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