CN107300594B - The method that liquid chromatography mass combination directly detects amino acid in biological tissue's Uniform Sample - Google Patents
The method that liquid chromatography mass combination directly detects amino acid in biological tissue's Uniform Sample Download PDFInfo
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- CN107300594B CN107300594B CN201710639232.5A CN201710639232A CN107300594B CN 107300594 B CN107300594 B CN 107300594B CN 201710639232 A CN201710639232 A CN 201710639232A CN 107300594 B CN107300594 B CN 107300594B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The invention discloses the methods that a kind of combination of liquid chromatography mass directly detects amino acid in biological tissue's Uniform Sample, including handling sample with extracting solution, the extracting solution by liquid chromatogram flowing phase composition, including acetonitrile and buffer, the concentration of acetonitrile is 50%-95%, the ammonium formate that the formic acid and concentration that buffer is 0.1%-0.3% by concentration are 1-10mM forms, or the ammonium acetate that the acetic acid and concentration for being 0.1%-0.3% by concentration are 1-10mM forms, and the pH of the extracting solution is between 3-5.5.Method disclosed by the invention can directly detect the biological tissues such as blood, urine and cerebrospinal fluid Uniform Sample, sample processing method is simple, sample dosage is few, can be down to 4 μ l, and detection sensitivity is high, up to pg/ml grades, detectable amino acid concentration range is wide, and matrix interference is small, and accuracy in detection is high, up to 29 kinds of the amino acid classes that can be detected simultaneously, and detection time is short.
Description
Technical field
The present patent application belongs to instrument analysis field, and in particular to a kind of liquid chromatography mass combination directly detection biology
The method of amino acid in even tissue sample.
Background technique
The detection and analysis of amino acid are the important research means in the fields such as life science, Food Science, clinical medicine, therefore
Have great importance to the research and improvement of amino acid analysis method.Amino acid analysis method was realized automatic first from 1958
Change, then gradually develops, sensitivity, accuracy, the degree of automation are all being gradually increased.
UPLC and Accqtag (Waters company) On-chip derivatization method are combined by Boogers in 2008 et al., analyze junket egg
The amino acid hydrolyzed in white and seralbumin.
Accqtag method needs to buy reagent packet and examination as a kind of current widely used amino acid analysis method
Agent box, higher cost, amino acid needs perform the derivatization, and the operation before experiment is relatively cumbersome, and derivating agent can only save one week,
Testing cost is high, and the amino acid of facile hydrolysis can not measure, and amino acid classes that can be detected are limited.
Biological tissue types are more, its physical property of different biological tissues, chemical group Chengdu are not quite similar, correspondingly,
The measurement method of amino acid composition content is also not quite similar.
Summary of the invention
At least for one of problems described above, the present invention provides directly detect biological group with liquid chromatography mass combination
The method for knitting amino acid in Uniform Sample, this method include handling biological tissue's Uniform Sample, the extracting solution packet with extracting solution
Include the mobile phase of liquid chromatogram.
As alternative embodiment of the present invention, extracting solution may include:
Acetonitrile, concentration 50%-95%；
Buffer, the ammonium formate that the formic acid and concentration for being 0.1%-0.3% by concentration are 1-10mM form, or by concentration
The ammonium acetate composition that acetic acid and concentration for 0.1%-0.3% are 1-10mM；
The pH of the extracting solution is between 3-5.5.
Alternative embodiment is disclosed as the present invention, and the preparation method of extracting solution may include:
(a) acetonitrile, formic acid or acetic acid, ammonium formate or ammonium acetate are mixed according to the volume ratio of 100:0.1:0.2, ultrasound is mixed
It is even, obtain organic phase；
(b) water and formic acid or acetic acid are mixed according to the volume ratio of 100:0.1, ultrasound mixes, and obtains water phase；
(c) it by obtained organic phase and water phase, is mixed according to the volume ratio of 5-6:1, ultrasound mixes, and obtains extracting solution.
Alternative embodiment is disclosed as the present invention, and extracting solution can be identical as liquid chromatogram initial flow phase composition.
Alternative embodiment is disclosed as the present invention, and biological tissue's Uniform Sample may include serum, urine or cerebrospinal fluid.
Alternative embodiment is disclosed as the present invention, and the processing method of biological tissue's Uniform Sample may include:
(i) -80 DEG C of refrigerator freezings are put samples into；
(ii) extracting solution is added according to the volume ratio 4:991 of sample and extracting solution in the sample after freezing；
(iii) the deuterated phenylalanine of internal standard is added, vortex mixed, the deuterated phenylalanine of internal standard is in final sample to be tested solution
In volume ratio be 0.5%；
(iv) 12000rpm, 4 DEG C of centrifugations, take supernatant, to be measured.
Further, alternative embodiment is disclosed as the present invention, the pH of extracting solution can be between 4.0-4.8.
Alternative embodiment is disclosed as the present invention, and the method for directly detecting amino acid in biological tissue's Uniform Sample further includes
Following steps:
A) amino acid standard mother liquor；
B) mother and sons' ion pair, orifice potential and collision energy is carried out to amino acid standard to grope；
C) liquid chromatography mass combination condition and Mass Spectrometry Conditions optimization；
D) amino acid standard curve is acquired；
E) amino acid content in liquid chromatography mass combination detection biological sample.
Alternative embodiment is disclosed as the present invention, and liquid chromatography mass combination condition may include:
Waters ACQUITY UPLC system, including quaternary pump solvent system, on-line degassing machine and autosampler；
XEVO TQ-S detector, MassLynxTM V4.1 mass spectrum workstation software；
Chromatographic column: ACQUITY UPLC BEH Amide, specification: 100mm × 2.1mm, 1.7 μm；
Mobile phase: A: acetonitrile contains 1mmolL-1Ammonium formate and 0.2% formic acid,
B: ultrapure water contains 1mmolL-1Ammonium formate and 0.1% formic acid；
Gradient elution:
Flow velocity: 0.3mLmin-1, column temperature: 40 DEG C, sample volume: 5 μ L.
Alternative embodiment is disclosed as the present invention, and Mass Spectrometry Conditions may include:
ESI ion source: positive ion mode；Capillary voltage: 0.4KV；Remove solvent temperature: 400 DEG C；Go solvent stream fast:
800L/Hr；Taper hole gas velocity: 150L/hr；Atomization gas pressure: 7.0Bar；It is acquired using MRM mode.
As the present invention, alternative embodiment is disclosed, when detecting serum sample amino acid, the preparation of amino acid standard mother liquor
It may include: that 100 μ gmL are formulated as with the aqueous solution containing 0.1% formic acid to L-2- aminoadipic acid and folic acid-1Standard
Product mother liquor；To remaining amino acid, 1mgmL is configured to ultrapure water-1Standard items mother liquor；Then dilute with extracting solution as needed
It releases, prepares hybrid standard product.
Alternative embodiment is disclosed as the present invention, and when detecting urine sample amino acid, the preparation of amino acid standard mother liquor can
To include: to be formulated as 100 μ gmL respectively with the aqueous solution containing 0.1% formic acid to homocysteine and L-2 aminoadipic acid-1、
1mg·mL-1Standard items mother liquor；To remaining amino acid, adding ultrapure water to be configured to concentration respectively is 1mgmL-1Standard items it is female
Liquid；Extracting solution dilution standard product mother liquor is used as needed, prepares hybrid standard product.
Alternative embodiment is disclosed as the present invention, and when detecting cerebrospinal fluid amino acid, the preparation of amino acid standard mother liquor can
To include: to be formulated as 100 μ gmL respectively with the aqueous solution containing 0.1% formic acid to homocysteine, L-2 aminoadipic acid-1、
1mg·mL-1Standard items mother liquor；To remaining amino acid standard, adding ultrapure water to be configured to concentration respectively is 1mgmL-1Mark
Quasi- product mother liquor；Extracting solution dilution standard product mother liquor is used as needed, prepares hybrid standard product.
Another aspect of the present invention provides a kind of in liquid chromatography mass combination directly detection biological tissue's Uniform Sample
The reagent packet of the method for amino acid, may include following component:
Acetonitrile, concentration 50%-95%；
Buffer, the ammonium formate that the formic acid and concentration that the buffer is 0.1%-0.3% by concentration are 1-10mM form,
Or the ammonium acetate that the acetic acid and concentration for by concentration being 0.1%-0.3% are 1-10mM forms；
The pH of the extracting solution is between 3-5.5.
The present invention discloses the method for being combined amino acid in directly detection biological tissue's Uniform Sample with liquid chromatography mass, can
Directly to detect after being handled with extracting solution biological samples such as serum, urine and cerebrospinal fluid, sample processing method is simple, sample
Dosage is few, and down to 4 μ l, detection sensitivity is high, and up to pg/ml grades, detectable amino acid concentration range is wide, the isotope of selection
Internal standard reduces matrix interference, improves accuracy in detection, while up to 29 kinds of amino acid classes detected, and detection time is short.
Detailed description of the invention
26 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 1 rat blood serum.
26 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 2 rat blood serum.
25 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 3 rat urine.
25 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 4 rat urine.
29 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 5 cerebrospinal fluid of rats.
29 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 6 cerebrospinal fluid of rats.
Specific embodiment
Biological sample is varied, have blood plasma, serum, whole blood, lymph, saliva, various tissues, hair, urine, bile,
Tear, spinal fluid, sweat, milk, amniotic fluid, excrement and the gas of exhalation.In the present disclosure, for the ease of ammonia in sample
The analysis of base sour component, mainly for liquid such as blood, urine, saliva, bile, cerebrospinal fluid, lymph, milk and sexual gland juices
Body tissue, this class loading can uniformly be classified as uniform biological tissue.It may be collectively referred to as the uniform sample of biological tissue in the present invention
This.
In the present disclosure, for the detection of biological tissue's Uniform Sample, the treatment process of biological organization sample is close,
To obtained sample to be tested, its detection method is similar, can be used as same class method and is investigated.
The present invention discloses the middle typical sample for selecting serum, urine and cerebrospinal fluid as uniform biological tissue, carries out amino acid
The detection of content.
Amino acid is amphiphatic molecule, and negative ions can dissociate, and degree of dissociation is related with the pH value of solution, such as into the solution
When acid is added, zwitterionic-COO-Anion, it is mobile to cathode in the electric field, when alkalinity is added, zwitterionic-
NH3+Cation releases proton, its own becomes anion, mobile to anode in the electric field, and amino acid is in the solution
Isoelectric point is also influenced by pH value, so usually all by the way of acid solution is added, such as hydrochloric acid, phosphoric acid, extract biological sample
Amino acid in this.But because the present invention is disclosed using the method that acidic buffer solution is added, guarantee effectively to extract amino
Acid.Such as formic acid/Ammonium formate buffer, acetic acid/ammonium acetate buffer, and its acidity value pH is carried out preferred.The selection of pH value can
It selects between 3-5.5, more preferred pH is more highly preferred to pH between 4.0-4.8 between 3.3-5.1.Meanwhile containing amino acid
Extracting solution as sample to be tested, when carrying out chromatograph mass spectrum analysis, extracting solution can flow through chromatographic column and mass spectral analysis ionisation chamber, institute
Direct influence is generated with the meeting testing result that forms of extracting solution, the present invention discloses formic acid/Ammonium formate buffer of selection, second
The requirement that composition in combined gas chromatography mass spectrometry analysis is easy gasification can be satisfied in acid/ammonium acetate buffer, improves analysis precision,
In consideration of it, the present invention discloses more preferred formic acid/Ammonium formate buffer.Moreover, the present invention, which discloses to use, contains formic acid/ammonium formate
Acetonitrile solution as chromatography mobile phase, mobile phase identical with extracting solution constituent can further promote to give birth to
The separation and detection of amino acid in object tissue samples.
Further, in buffer buffer salt concentration, also have an impact to biological tissue extracted effect, such as ammonium formate or second
Sour ammonium can choose any concentration between 1-10mM.Concentration is lower than 1mM or is higher than 10mM, and extraction efficiency is decreased obviously,
The width of chromatographic peak obviously broadens, and is unfavorable for the extraction and separation of amino acid.
The present invention discloses in implementation, internal standard use deuterated phenylalanine standard items, process for preparation may include take it is deuterated
Phenylalanine standard product, accurately weighed, being configured to concentration with ultrapure water is 1mgmL-1Internal standard mother liquor, then as needed use
Extracting solution is diluted.For example, can be by 100 μ L, 1mgmL-1Internal standard mother liquor mixed with 900 μ L extracting solutions, be vortexed mix,
It is configured to 10 μ gmL-1Inner mark solution, can be by 10 μ L, 10 μ gmL-1Inner mark solution mixed with 990 μ L extracting solutions,
It is vortexed and mixes, be configured to 100ngmL-1Inner mark solution.
In the preparation or preparation method of extracting solution disclosed by the invention, it is slow for illustratively describing with formic acid and ammonium formate
The specific implementation of fliud flushing component meets the buffer of open request of the present invention as other, such as acetic acid and ammonium acetate buffer
System or other buffer solution systems can also use same method, extract the preparation of liquid.
Further, as extracting solution disclosed by the invention, the content of acetonitrile directly affects extraction efficiency and detection sensitivity,
For example, can choose its content between 50%-95%, perhaps between 60-90% or can be between 70-85%.
As the selection of extracting solution, can be carried out according to the property of biological organization sample preferred.For example, can be formulated as follows
The extracting solution of component:
(1) 50% acetonitrile solution includes 0.1% formic acid and 1mmol ammonium formate, pH 3.18；
(2) 85% acetonitrile solutions include 0.1% formic acid and 1.7mmol ammonium formate, pH 4.32；
(3) 95% acetonitrile solutions include 0.1% formic acid and 1.9mmol ammonium formate, pH 5.46；
(4) 85% acetonitrile solutions include 0.1% formic acid and 10mmol ammonium formate, pH 5.11；
Ammonia by the Performance discovery to above four kinds of extracting solutions, after the processing of the above extracting liq, in sample
Base acid can be extracted efficiently, only peak shape and respective strengths different from, for example, result is wanted in extracting solution (2) and (4)
Better than (1) and (3), wherein the result in (2) is more excellent.
The quantity of biological organization sample can also have an impact method disclosed by the invention, generally biological organization sample
In the detection of middle amino acid, matrix effect can all generate bigger influence to testing result, and during the present invention discloses, selection is appropriate
Sample size, then the influence of matrix effect can be effectively controlled, for example, biological sample can eliminate base between 4-10 μ L
Influence of the mass effect to testing result.In view of sample processing method disclosed by the invention and analysis method, biological sample is more excellent
It selects between 4-8 μ L, between more preferable 4-6 μ L.
The quantity of extracting solution also has an impact the extraction of amino acid in biological organization sample.Because biological sample matrix
Complexity, the volume for choosing biological sample is smaller, and the volume of extracting solution is bigger, and the influence of matrix effect is smaller, such as the present invention
In open, the volume of extracting solution is 250 times of biological organization sample, can effectively extract the ammonia in biological tissue within this range
Base acid constituents, and can satisfy the separation and detection demand after extracted to amino acid.
Flow phase constituent selection, the factor mainly considered first is that being convenient for during liquid chromatogram, mass spectrum separation detection
The separation and detection of amino acid improve detection sensitivity and resolution ratio, and the flowing phase constituent for being easy to gasify is conducive to mass spectrum
Detection so mobile phase disclosed by the invention uses organic phase and aqueous phase solution containing buffer, such as contains formic acid/formic acid
The acetonitrile solution of ammonium buffer is water phase containing formic acid/Ammonium formate buffer ultrapure water as organic phase.For example, organic phase
For acetonitrile, wherein containing 1mmolL-1(mol·L-1It can be expressed as M, mmolL-1Can be expressed as mM) ammonium formate and 0.2%
Formic acid, water phase are ultrapure water, wherein containing 1mmolL-1Ammonium formate and 0.1% formic acid.
In detection method disclosed by the invention, the extracting solution of use and the mobile phase of liquid chromatogram can have identical group
At component, this not only contributes to the extraction of amino acid in biological organization sample, and is conducive in analysis phase different aminoacids
Efficiently separate and detect completely, moreover, as more preferred scheme, can choose that constituent component is identical, content is close
Extracting solution and mobile phase, it is more preferred to, the component and concentration of extracting solution and the concentration of component of liquid chromatogram initial liquid phase
When corresponding to consistent completely, there is more preferably analysis detection result.
Mother and sons' ion pair, orifice potential and collision energy is carried out to each amino acid standard to grope.By each amino acid from
Concentration is that 1ng/mL starts to be gradually increased concentration, is touched using the Intellistart function of UPLC/XEVO TQ-S instrument
Rope, until mother and sons' ion pair, orifice potential and the collision energy of each amino acid are confirmed, such as table 1.
Mother and sons' ion pair, orifice potential and collision energy of 1. 29 kinds of amino acid of table
Combined gas chromatography mass spectrometry condition and Mass Spectrometry Conditions are optimized.
The amino acid mothers and sons ion pair determined, orifice potential collision energy are incorporated into MRM mass spectrometry method, chromatographic column selection
ACQUITY UPLC BEH Amide, 1.7 μm, 2.1x100mm, from feeder, it is ensured that each normal appearance of amino acid.
In amino acid detection method disclosed by the invention, the processing step that the processing method of biological organization sample is related to,
There is no its processing sequences of considered critical, if not otherwise specified, or within the scope of testing allows, can be according to need
Adjustment appropriate is done to processing step, the processing result of sample is had no effect on, also within the scope of spirit of the invention.
The present invention disclose in the numerical parameter that is indicated with range or section, in the range of typically referring to including endpoints thereof
All numerical value.Unless specifically stated otherwise, it is the ratio between volume that the present invention, which discloses addressed percent concentration,.
In amino acid detection method disclosed by the invention, the processing method of biological organization sample can also include in order to just
In other steps that sample process needs, such as the packing of sample, the preparation or preformulation of extracting solution, the increase of these steps or
It reduces, not departing from the range of amino acid direct detecting method disclosed by the invention.
In amino acid detection method disclosed by the invention, each step that detection method includes, there is no considered critical its
Sequentially, if not otherwise specified or the logical requirements of detection method certainty, it can be done suitable according to actually detected demand
Work as adjustment, have no effect on testing result, and can according to need and increase other steps, such as increases repeatability and investigate step, return
Yield investigates step, also can according to need and reduces some steps, such as amino acid standard curve (or linear investigate) step or
It is real not affect spirit disclosed by the invention as long as the change that the needs of meeting amino acid detection is done for mother liquor step etc.
Matter.
The present invention discloses being combined for liquid chromatography mass for offer and directly detects amino acid in biological tissue's Uniform Sample
Reagent packet, can be the form of reagent packet, be also possible to kit or other facilitate the form of offer；At biological sample
The reagent of extracting solution is managed, offer form does not change its function used as extracting solution, so will not change in fact
Matter content, the variation of any other form, all within spirit disclosed by the invention.
During the present invention discloses, unless specifically stated otherwise, the technical term addressed have those skilled in the art understand that it is usual
Meaning.Liquid chromatogram and the analysis method of mass spectrometry refer to LC-MS/MS.
The disclosure of exemplary description and data by the following examples, the details and invention essence of technical solution of the present invention
It will be more clear, it is clear, it should be understood that these details are not the limitation to substantive content of the present invention and protection scope.
The detection of serum sample.
1. laboratory apparatus
2. reagent
Standard items: glycine (Glycine), Beta-alanine (β-Alanine), sarcosine (Sarcosine), alanine
(Alanine), γ-aminobutyric acid (γ-Aminobutyric acid), serine (Serine), valine (Valine), Soviet Union
Propylhomoserin (Threonine), taurine (Taurine), leucine (Leucine), isoleucine (Isoleucine), trans--hydroxyl
Proline (trans-4-Hydroxy-L-Proline), ornithine (Ornithine), relies asparagine (Asparaginate)
Propylhomoserin (Lysine), methionine (Methionine), L-2 aminoadipic acid (L-2-Aminoadipic acid), phenylpropyl alcohol ammonia
Acid (Phenylalanine), 1- methyl-histidine (1-Methyl-histidine), 3- methyl-histidine (3-Methyl-
Histidine), arginine (Arginine), citrulling (Citrulline), tryptophan (Tryptophan), folic acid (Folic
Acid it) is purchased from sigma company, the U.S., glutamine (Glutamine), histidine (Histidine) are purchased from Canadian TCI
Company.
Acetonitrile and formic acid: mass spectrum grade, Fisher company, the U.S.；Ammonium formate: sigma company, the U.S.；Experimental water: Milli-
The ultrapure water purification system preparation of Q system.
3. sample
Serum sample comes from Sprague-Dawley rat, according to biological tissue's Uniform Sample requirement of experiment, according to specification,
Reasonable operating process acquisition provides.
4. combined gas chromatography mass spectrometry condition
Waters ACQUITY UPLC system, including quaternary pump solvent system, on-line degassing machine and autosampler；
Waters company, Milford, USA；XEVO TQ-S detector, Waters company；MassLynxTMV4.1 mass spectrum work station is soft
Part, Waters company；Chromatographic column: ACQUITY UPLC BEH Amide, specification: 100mm × 2.1mm, 1.7 μm；
Mobile phase A: acetonitrile: contain 1mmolL-1Ammonium formate and 0.2% formic acid,
B: ultrapure water: contain 1mmolL-1Ammonium formate and 0.1% formic acid；
Gradient elution: 2 are shown in Table；
2 gradient elution time of table, the mobile phase table of comparisons
Flow velocity: 0.3mLmin-1, column temperature: 40 DEG C, sample volume: 5 μ L.
5. Mass Spectrometry Conditions
ESI ion source: positive ion mode；Capillary voltage: 0.4KV；Remove solvent temperature: 400 DEG C；Go solvent stream fast:
800L/Hr；Taper hole gas velocity: 150L/hr；Atomization gas pressure: 7.0Bar；It is acquired using MRM mode.
6. the preparation of standard items mother liquor
Take all amino acid standards appropriate, it is accurately weighed, to L-2 aminoadipic acid, folic acid, with containing 0.1% formic acid
Aqueous solution be formulated as 100 μ gmL respectively-1Standard items mother liquor add ultrapure water to be configured to concentration respectively in remaining amino acid
For 1mgmL-1Standard items mother liquor, be diluted as needed with extracting solution, and prepare hybrid standard product, it is big with S/N respectively
4 are shown in Table as detection limit (LOD) and quantitative limit (LOQ), LOD and LOQ concentration in 3,10, hybrid standard product spectrogram such as Fig. 1 serum
In 26 kinds of amino acid LC-MS/MS spectrum (standard items).
7. extracting solution is prepared
Organic phase: the ammonium formate mixing of 85mL acetonitrile, 85 μ L formic acid, 17 μ L10mol/L, ultrasound mix；
Water phase: 15mL ultrapure water, the mixing of 15 μ L formic acid, ultrasound mix；
After the two mixing, then ultrasound 3min is mixed, and obtains extracting solution.
8. sample preparation
Rat blood serum is distributed into L/ parts of 4 μ, -80 DEG C of refrigerator freezings.The serum dispensed is taken out, 991 μ L are added and extract
Liquid, is added 5 μ L internal standards (deuterated phenylalanine 100ng/mL), and 1min, 12000rpm, 4 DEG C of centrifugation 5min of vortex mixed take
Clear liquid is to be measured.Monitoring result is shown in 26 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 2 serum sample.
9. pattern detection
9.1 detection method linear relationships are investigated
3 rat blood serum sampled amino acid standard items of table
The preparation of linear mother liquor.Amino acid standard in extracting solution and table 3 is taken, 2mL mother liquor, vortex 1min are formulated as
(max), extracting solution is added to be diluted to the hybrid standard product solution of series of concentrations, the hybrid standard product solution of each concentration contains
The deuterated phenylalanine of 0.5ng/mL is calibrated as internal standard compound, and by set chromatographic condition and Mass Spectrometry Conditions, sample introduction is analyzed, with
The peak area ratio of each amino acid and internal standard compound is ordinate (Y), is that abscissa (X) carries out linear regression with concentration (pg/ μ L),
R is calculated, r value shows that this method is linearly good in 0.9811-0.9999, as a result such as the following table 4.
The linear equation of 26 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 4. rat blood serum sample of table
9.2 precision and repeatability are investigated
Withinday precision is investigated: choose low (L), in (M), each 1 part of hybrid standard product solution of high (H) point concentration, continuously
Sample introduction 6 times measurements, as shown in table 5 below, the RSD (%) of 26 kinds of amino acid peak areas shows this method between 1.27-36.7%
Withinday precision it is good.
Day to day precision is investigated: choose low (L), in (M), each 1 part of hybrid standard product solution of high (H) point concentration, continuous 3
The measurement of its sample introduction, as shown in table 5 below, the RSD (%) of 26 kinds of amino acid peak areas shows this method between 1.13-36.09%
Day to day precision it is good.
Repeatability is investigated: 6 parts of sample with a batch packing taken, prepares sample to be tested solution by aforementioned sample preparation methods,
It is measured.As a result as shown in table 5 below, the RSD (%) of 26 kinds of amino acid peak areas shows we between 0.70-8.81%
The repeatability of method is good.
The day to day precision of 26 kinds of amino acid, withinday precision and method repeatability in 5. rat blood serum sample of table
9.3 the investigation of the rate of recovery
Take 18 parts of sample dispensed, be divided into 3 groups, 4 DEG C of refrigerator defrostings, be separately added into concentration of specimens 50%, 100%,
150% standard solution prepares sample to be tested solution, is measured, rate of recovery accounting equation: the rate of recovery (%)=(measurement
Value-sample content)/additional amount × 100%, as shown in table 6 below, the rate of recovery exists the RSD (%) of the rate of recovery and its peak area
72.71%-128.62% shows that this method is accurate and reliable.
The rate of recovery of 26 kinds of amino acid in 6. rat blood serum sample of table
9.4 freeze-thaw stabilities are investigated
12 parts of sample dispensed are taken, are divided into 3 groups, every group of sample number n is 4, freeze thawing 3 times respectively, each 12h, by sample
Processing method prepares sample to be tested solution, is measured, and the results are shown in Table 7, the RSD (%) of peak area is in 3.24%-10.92%
Between, show to stablize by 3 freeze thawing samples.
The freeze-thaw stability of 26 kinds of amino acid in 7. rat blood serum sample of table
The detection of urine sample.
1. laboratory apparatus
With embodiment 1.
2. reagent
Glycine (Glycine), Beta-alanine (β-Alanine), sarcosine (Sarcosine), alanine
(Alanine), γ-aminobutyric acid (γ-Aminobutyric acid), serine (Serine), histamine (Histamine), figured silk fabrics
Propylhomoserin (Valine), taurine (Taurine), leucine (Leucine), isoleucine (Isoleucine), trans--hydroxyl dried meat ammonia
Sour (trans-4-Hydroxy-L-Proline), asparagine (Asparaginate), ornithine (Ornithine), height half
Cystine (Homocysteine), lysine (Lysine), glutamic acid (Glutamic Acid), methionine
(Methionine), L-2 aminoadipic acid (L-2-Aminoadipic acid), phenylalanine (Phenylalanine), essence
Propylhomoserin (Arginine), citrulling (Citrulline), tryptophan (Tryptophan), folic acid (Folic acid) are purchased from beauty
Sigma company, state, glutamine (Glutamine) are purchased from Canadian TCI company.
Acetonitrile and formic acid: mass spectrum grade, Fisher company, the U.S.；Ammonium formate: sigma company, the U.S.；Experimental water: Milli-
The ultrapure water purification system preparation of Q system.
3. sample
Urine sample comes from Sprague-Dawley rat, specification, the reasonable operation stream according to biological tissue's Uniform Sample
Journey acquisition provides.
4. combined gas chromatography mass spectrometry condition
With embodiment 1.
5. Mass Spectrometry Conditions
With embodiment 1.
6. the preparation of standard items mother liquor
Take all amino acid standards appropriate, it is accurately weighed, to homocysteine, L-2 aminoadipic acid, with containing
The aqueous solution of 0.1% formic acid is formulated as 100 μ gmL respectively-1、1mg·mL-1Standard items mother liquor remaining amino acid is added super
It is 1mgmL that pure water is configured to concentration respectively-1Standard items mother liquor, be diluted as needed with extracting solution, and prepare mixing
Standard items are greater than 3,10 as detection limit (LOD) and quantitative limit (LOQ) using S/N respectively, and it is dense that LOD and LOQ concentration is shown in Table 8, LOD
For degree in 0.0036-10ng/mL, LOQ concentration shows that this method sensitivity is very high in 0.016-20ng/mL.Hybrid standard product spectrogram
Such as 25 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 3 rat urine.
7. extracting solution is prepared
With embodiment 1.
8. sample preparation
Urine sample is centrifuged, 12000rpm, 4 DEG C, 5min takes supernatant spare.It takes 991 μ g extracting solutions, is added 4 μ L urine samples, 5
μ L internal standard (is inside designated as deuterated benzene alanine, concentration 100ng/mL), 1min, 12000rpm, 4 DEG C of centrifugation 5min of vortex mixed,
Take supernatant to be measured.
Testing result is shown in 25 kinds of amino acid LC-MS/MS spectrums in Fig. 4 rat urine sample.
9. pattern detection
9.1 linear relationships are investigated
9 rat urine sampled amino acid standard items of table
The preparation of linear mother liquor.Urine sample amino acid standard in extracting solution and table 9 is taken, 2mL mother liquor is formulated as, is vortexed
1min (max), adds extracting solution to be diluted to the hybrid standard product solution of series of concentrations, and the hybrid standard product solution of each concentration is equal
Deuterated phenylalanine containing 0.5ng/mL is calibrated as internal standard compound, and by set chromatographic condition and Mass Spectrometry Conditions, sample introduction is analyzed,
As a result, it has been found that urine sample matrix internally indicates inhibiting effect, therefore processing method does not select internal standard, is vertical sit with each amino acid peak area
It marks (Y), is that abscissa (X) carries out linear regression with concentration (pg/ μ L), calculates r, r value shows between 0.9858-0.9999
This method is linearly good.As a result such as table 8.
The linear equation of 25 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 8. rat urine sample of table
9.2 precision and repeatability are investigated
Withinday precision is investigated: each 1 part of hybrid standard product solution for choosing basic, normal, high concentration, continuous sample introduction 6 times surveys
Fixed, as shown in the following table 10, the RSD (%) of 25 kinds of amino acid peak areas shows the day of this method between 1.24%-20.23%
Interior precision is good.
Day to day precision is investigated: each 1 part of hybrid standard product solution for choosing basic, normal, high concentration, sample introduction is surveyed for three days on end
Fixed, the RSD of 25 kinds of amino acid peak areas is as shown in the following table 10, and the RSD (%) of amino acid is between 1.14%-38.60%, table
Bright this method day to day precision is good.
Repeatability is investigated: 6 parts of sample with a batch packing taken, prepares sample to be tested solution by aforementioned sample preparation methods,
It is measured.As a result as shown in the following table 10, the RSD (%) of peak area shows that the repeatability of this method is good in 1.31-9.60%.
The day to day precision of 25 kinds of amino acid, withinday precision and method repeatability in 10. rat urine sample of table
The investigation of 9.3 rate of recovery
Take 18 parts of sample dispensed, be divided into 3 groups, 4 DEG C of refrigerator defrostings, be separately added into concentration of specimens 50%, 100%,
50%, 100%, 150% standard solution of LOQ concentration is added in 150% standard solution, undetected amino acid,
Sample to be tested solution is prepared, is measured, rate of recovery accounting equation: the rate of recovery (%)=(measured amount-background)/additional amount ×
100%, the RSD of the rate of recovery and its peak area is as shown in table 11 below, and the rate of recovery (%) shows between 72.12%-126.63%
This method is accurate and reliable.
The rate of recovery of 25 kinds of amino acid in 11. rat urine sample of table
9.4 freeze-thaw stabilities are investigated
12 parts of sample dispensed are taken, are divided into 3 groups, every group of sample number n is 4, freeze thawing 3 times respectively, each 12h, by preceding sample
Treatment method prepares sample to be tested solution, is measured, and the results are shown in Table 12, RSD (%) between 2.52%-9.82%, table
Bright to pass through 3 freeze thawing, sample is stablized.
The freeze-thaw stability of 25 kinds of amino acid in 12. rat urine sample of table
CSF sample detection
1. laboratory apparatus
With embodiment 1.
2. reagent
Standard items: glycine (Glycine), Beta-alanine (β-Alanine), sarcosine (Sarcosine), alanine
(Alanine), γ-aminobutyric acid (γ-Aminobutyric acid), serine (Serine), histamine (Histamine), figured silk fabrics
Propylhomoserin (Valine), threonine (Threonine), taurine (Taurine), leucine (Leucine), isoleucine
(Isoleucine), trans--hydroxyproline (trans-4-Hydroxy-L-Proline), asparagine (Asparaginate),
Ornithine (Ornithine), homocysteine (Homocysteine), lysine (Lysine), glutamic acid (Glutamic
Acid), methionine (Methionine), L-2 aminoadipic acid (L-2-Aminoadipic acid), phenylalanine
(Phenylalanine), 1- methyl-histidine (1-Methyl-histidine), 3- methyl-histidine (3-Methyl-
Histidine), arginine (Arginine), citrulling (Citrulline), tryptophan (Tryptophan), carnosine
(Carnosine) it is purchased from sigma company, the U.S., glutamine (Glutamine), histidine (Histidine) are purchased from plus take
Big TCI company.
Acetonitrile and formic acid: mass spectrum grade (Fisher company, the U.S.)；Ammonium formate: sigma company, the U.S.；Experimental water:
The ultrapure water purification system preparation of Milli-Q system.
3. sample
CSF sample comes from Sprague-Dawley rat, according to uniform biological sample requirement of experiment, according to specification, closes
The operating process of reason, which acquires, to be provided.
4. combined gas chromatography mass spectrometry condition
With embodiment 1.
5. Mass Spectrometry Conditions
With embodiment 1.
6. the preparation of standard items mother liquor:
Appropriate amino acid standard is taken, it is accurately weighed；To homocysteine, L-2 aminoadipic acid, with containing 0.1% first
The aqueous solution of acid is formulated as 100 μ gmL respectively-1、1mg·mL-1Standard items mother liquor；To remaining amino acid standard, add super
It is 1mgmL that pure water is configured to concentration respectively-1Standard items mother liquor；It is diluted as needed with extracting solution, and prepares mixing
Standard items；It is greater than 3,10 as detection limit (LOD) and quantitative limit (LOQ), LOD and LOQ concentration using S/N respectively and is shown in Table 14, LOD
Concentration shows that this method sensitivity is very high in 0.016-20ng/mL in 0.0036-10ng/mL, LOQ concentration.Hybrid standard product spectrum
Figure such as 29 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 5 cerebrospinal fluid of rats.
7. extracting solution is prepared
With embodiment 1.
8. sample preparation
Cerebrospinal fluid sample is distributed into L/ parts of 4 μ, -80 DEG C of refrigerator freezings.The cerebrospinal fluid dispensed is taken out, 991 μ L are added and mention
It takes liquid, is added 5 μ L internal standards (being inside designated as deuterated benzene alanine, concentration 100ng/mL), vortex mixed 1min, 12000rpm,
4 DEG C of centrifugation 5min, take supernatant to be measured.Testing result is shown in 29 kinds of amino acid LC-MS/MS spectrum (samples in Fig. 6 cerebrospinal fluid of rats
This).
9. pattern detection
9.1 linear relationships are investigated
13 cerebrospinal fluid of rats sampled amino acid standard items of table
The preparation of linear mother liquor.Amino acid standard listed by extracting solution and upper table 13 is taken, 2mL mother liquor is formulated as, is vortexed
1min (max), adds extracting solution to be diluted to the hybrid standard product solution of series of concentrations, and the hybrid standard product solution of each concentration is equal
Deuterated phenylalanine containing 1.5ng/mL is calibrated as internal standard compound, and by set chromatographic condition and Mass Spectrometry Conditions, sample introduction is analyzed,
It is that abscissa (X) is linearly returned with concentration (pg/ μ L) using the peak area ratio of each amino acid and internal standard compound as ordinate (Y)
Return, calculate r, r value shows that this method is linearly good in 0.9811-0.9999.As a result as 29 kinds in 14 cerebrospinal fluid of rats sample of table
Linear equation, the range of linearity and the detection limit and quantitative limit of amino acid.
The linear equation of 29 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 14. cerebrospinal fluid of rats sample of table
9.2 precision and repeatability are investigated
Withinday precision is investigated: each 1 part of hybrid standard product solution for choosing basic, normal, high concentration, continuous sample introduction 6 times surveys
Fixed, the RSD (%) of 29 kinds of amino acid peak areas as shown in table 15 below, between 1.02%-22.95%, shows the day of this method
Interior precision is good.
Day to day precision is investigated: each 1 part of hybrid standard product solution for choosing basic, normal, high concentration, sample introduction is surveyed for three days on end
Fixed, the RSD (%) of 29 kinds of amino acid peak areas as shown in table 15 below, between 0.61%-22.56%, shows our France and Japan
Between precision it is good.
Repeatability is investigated: 6 parts of sample with a batch packing taken, prepares test solution by aforementioned sample preparation methods, into
Row measurement.As a result as shown in table 15 below, the RSD (%) of 29 kinds of amino acid peak areas shows this between 2.29%-7.50%
The repeatability of method is good.
The day to day precision of 29 kinds of amino acid, withinday precision and method repeatability in 15 cerebrospinal fluid of rats sample of table
The investigation of 9.3 rate of recovery
Take 18 parts of sample dispensed, be divided into 3 groups, 4 DEG C of refrigerator defrostings, be separately added into concentration of specimens 50%, 100%,
150% standard solution prepares sample to be tested solution, is measured, rate of recovery accounting equation: the rate of recovery (%)=(measure
Amount-sample content)/additional amount × 100%, as shown in table 16 below, the rate of recovery exists the RSD (%) of the rate of recovery and its peak area
70.40%-126.98% shows that this method is accurate and reliable.
The rate of recovery of 29 kinds of amino acid in 16. cerebrospinal fluid of rats sample of table
9.4 freeze-thaw stabilities are investigated
12 parts of sample dispensed are taken, are divided into 3 groups, every group of sample number n is 4, freeze thawing 3 times respectively, each 12h, by preceding sample
Treatment method prepares sample to be tested solution, is measured, and the results are shown in Table 17, the RSD (%) of peak area is in 2.47%-
6.92%, show that, by 3 freeze thawing, sample is stablized.
The freeze-thaw stability of 29 kinds of amino acid in 17. cerebrospinal fluid of rats sample of table
Claims (3)
1. the method that liquid chromatography mass combination directly detects amino acid in biological tissue's Uniform Sample characterized by comprising
Biological tissue's Uniform Sample is handled with extracting solution；
Amino acid standard is prepared；
Wherein, biological tissue's Uniform Sample includes serum, urine and cerebrospinal fluid；
It is described to be specifically included with extracting solution processing biological tissue's Uniform Sample:
(i) -80 DEG C of refrigerator freezings are put samples into；
(ii) the sample 4-6 μ L after freezing is mixed according to the volume ratio 4:991 of sample and extracting solution with extracting solution；
(iii) the deuterated phenylalanine of internal standard is added, vortex mixed, the deuterated phenylalanine of internal standard is in final sample to be tested solution
Volume ratio is 0.5%；
(iv) 12000rpm, 4 DEG C of centrifugations, take supernatant, to be measured；
The amino acid standard is prepared
(a) serum sample amino acid detects
To L-2- aminoadipic acid and folic acid, 100 μ gmL are formulated as with the aqueous solution containing 0.1% formic acid-1Standard items it is female
Liquid；
To remaining amino acid, 1mgmL is configured to ultrapure water-1Standard items mother liquor；
With extracting solution dilution standard product mother liquor, hybrid standard product are prepared；
(b) urine sample and the detection of CSF sample amino acid
To homocysteine and L-2 aminoadipic acid, it is formulated as 100 μ gmL respectively with the aqueous solution containing 0.1% formic acid-1、
1mg·mL-1Standard items mother liquor；
To remaining amino acid, adding ultrapure water to be configured to concentration respectively is 1mgmL-1Standard items mother liquor；
With extracting solution dilution standard product mother liquor, hybrid standard product are prepared；
The extracting solution includes:
Acetonitrile, concentration 85%；
Buffer, the ammonium formate that the formic acid and concentration for being 0.1% by concentration are 1.7mM form；
The pH of the extracting solution is 4.32.
2. according to the method described in claim 1, further comprising the steps of:
A) mother and sons' ion pair, orifice potential and collision energy is carried out to amino acid standard to grope；
B) liquid chromatography mass combination condition and Mass Spectrometry Conditions optimization；
C) amino acid standard curve is acquired；
D) amino acid content in liquid chromatography mass combination detection biological sample.
3. according to the method described in claim 2, it is characterized in that, the liquid chromatography mass combination condition includes:
Waters ACQUITYUPLC system, including quaternary pump solvent system, on-line degassing machine and autosampler；
XEVO TQ-S detector, MassLynxTM V4.1 mass spectrum workstation software；
Chromatographic column: ACQUITY UPLC BEHAmide, specification: 100mm × 2.1mm, 1.7 μm；
Mobile phase A: acetonitrile contains 1mmolL-1Ammonium formate and 0.2% formic acid,
B: ultrapure water contains 1mmolL-1Ammonium formate and 0.1% formic acid；
Gradient elution:
Flow velocity: 0.3mLmin-1, column temperature: 40 DEG C, sample volume: 5 μ L；
The Mass Spectrometry Conditions include:
ESI ion source: positive ion mode；Capillary voltage: 0.4KV；Remove solvent temperature: 400 DEG C；Go solvent stream fast: 800L/
Hr；Taper hole gas velocity: 150L/hr；Atomization gas pressure: 7.0Bar；It is acquired using MRM mode.
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| CN107843672B (en) * | 2017-12-21 | 2020-09-08 | 上海中科新生命生物科技有限公司 | Method for detecting amino acid in serum by high performance liquid chromatography-tandem mass spectrometry |
| CN108333268A (en) * | 2018-01-30 | 2018-07-27 | 济南英盛生物技术有限公司 | Method that is a kind of while detecting 40 kinds of amino acid in dry blood cake, blood and urine |
| CN109856258A (en) * | 2018-12-29 | 2019-06-07 | 中山百灵生物技术有限公司 | A kind of detection method of new content of taurine |
| CN109633030A (en) * | 2019-01-22 | 2019-04-16 | 江苏澳华生物科技研究院有限公司 | A kind of method that ultra performance liquid chromatography-QQ-TOF mass spectrometry detects amino acid in animal body fluid or tissue samples |
| CN111879859B (en) * | 2019-11-27 | 2021-10-08 | 江南大学 | Method for accurately detecting content of butanediamine in fermentation liquor |
| CN110806458A (en) * | 2019-11-29 | 2020-02-18 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting leucine, isoleucine and valine contents in blood |
| CN112162049B (en) * | 2020-10-13 | 2022-12-02 | 合肥谱佳医学检验实验室有限公司 | Method for detecting sarcosine in urine for non-diagnosis purpose |
| WO2022120752A1 (en) * | 2020-12-10 | 2022-06-16 | 中国科学院深圳先进技术研究院 | Method for quantitative analysis of free amino acids in biological sample by liquid chromatography-tandem mass spectrometry |
| CN113009030A (en) * | 2021-03-01 | 2021-06-22 | 上海阿趣生物科技有限公司 | Amino acid high-throughput target detection method and application thereof |
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