CN107192783B - The method that liquid chromatography mass combination directly detects amino acid in biological tissue's nonuniform sample - Google Patents
The method that liquid chromatography mass combination directly detects amino acid in biological tissue's nonuniform sample Download PDFInfo
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- CN107192783B CN107192783B CN201710639231.0A CN201710639231A CN107192783B CN 107192783 B CN107192783 B CN 107192783B CN 201710639231 A CN201710639231 A CN 201710639231A CN 107192783 B CN107192783 B CN 107192783B
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- G—PHYSICS
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- 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 nonuniform 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 by concentration being 0.1%-0.3% are 1-10mM forms, the pH of extracting solution is between 3-5.5.Method disclosed by the invention can directly detect the biological tissues such as cardiac muscular tissue, cerebral cortex and hippocampal tissue nonuniform 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 for organizing amino acid in nonuniform 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,
The amino acid of facile hydrolysis can not measure, and the amino acid classes being able to detect that 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 nonuniform sample, this method include handling biological tissue's nonuniform sample, the extraction with extracting solution
Liquid includes the mobile phase of liquid chromatogram.
Alternative embodiment is disclosed as the present invention, and 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, ammonium formate are mixed according to the volume ratio of 100:0.1:0.2, ultrasound mixes, and obtains organic phase；
(b) water and formic 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 nonuniform sample can be cardiac muscular tissue, cerebral cortex group
It knits or hippocampal tissue.
Alternative embodiment is disclosed as the present invention, and the processing method of biological tissue's nonuniform sample may include:
(i) biological tissue is cleaned with physiological saline, is dried, shredded and mixed；
(ii) it weighs, adds 20% methanol to be homogenized, obtain homogenate；
(iii) homogenate is put into -80 DEG C of refrigerator freezings；
(iv) extracting solution is added according to the volume ratio 4:991 of homogenate and extracting solution in the homogenate of freezing；
(v) the deuterated phenylalanine of internal standard, vortex mixed, body of the deuterated phenylalanine of internal standard in final solution to be measured is added
Product content is 0.5%；
(vi) 12000rpm, 4 DEG C of centrifugations, take supernatant, to be measured.
Alternative embodiment is disclosed as the present invention, and the pH of extracting solution can be between 4.0-4.8.
As the present invention, alternative embodiment is disclosed, the method for amino acid directly in detection biological tissue's nonuniform sample, also
It may comprise steps of:
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 cardiac muscular tissue's amino acid, the preparation of amino acid standard mother liquor
It may include: to be formulated as respectively to homocysteine, L-2- aminoadipic acid and tyrosine with the aqueous solution containing 0.1% formic acid
100μg·mL-1、1mg·mL-1、250μg·mL-1Standard items mother liquor；To remaining amino acid, 1mg/mL is configured to ultrapure water
Standard items mother liquor；Then extracting solution dilution standard product mother liquor is used as needed, is configured to hybrid standard product.
Disclose alternative embodiment as the present invention, when detection cortical tissue's amino acid, the preparation of amino acid standard can be with
It include: that 100 μ gmL are formulated as respectively with the aqueous solution containing 0.1% formic acid to homocysteine, tyrosine, folic acid-1、
250μg·mL-1、100μg·mL-1Standard items mother liquor；To remaining amino acid, add ultrapure water to be configured to concentration respectively to be
1mg·mL-1Standard items mother liquor；Extracting solution dilution standard product mother liquor is used as needed, and prepares hybrid standard product.
Disclose alternative embodiment as the present invention, when detection hippocampal tissue amino acid, the preparation of amino acid standard can be with
It include: that 100 μ gmL are formulated as 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, and prepares hybrid standard product.
The present invention, which discloses, also provides a kind of be used in liquid chromatography mass combination directly detection biological tissue's nonuniform sample
The reagent packet of 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 nonuniform sample with liquid chromatography mass,
It is directly detected after being handled with extracting solution biological samples such as cardiac muscular tissue, cerebral cortex and hippocampal tissues, at sample
Reason method is simple, and sample dosage is few, and down to 4 μ l, detection sensitivity is high, pg/ml grades reachable, detectable amino acid concentration range
The Isotopic Internal Standard of width, selection 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
29 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 1 rat heart muscle tissue.
29 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 2 rat heart muscle tissue.
28 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 3 rat cortical tissue.
28 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 4 rat cortical tissue.
20 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 5 Rat hippocampus.
20 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 6 Rat hippocampus.
Specific embodiment
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.
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 mainly for non-homogeneous sample
This progress amino acid detection, the heterogene structure include: the heart, liver, spleen, lung, kidney, stomach, intestines, reproductive organs, brain, body fat, chest
The organs such as gland adrenal gland and skeletal muscle, tissue.
In the present disclosure, for nonuniform sample, representative cardiac muscular tissue, cerebral cortex group are mainly used
It knits and hippocampal tissue, carries out the measurement of amino acid content.Detection for biological tissue's nonuniform sample, biological organization sample
Treatment process is close, similar to the detection method of amino acid in obtained sample to be tested, can be used as same class method and is examined
It examines.
It only needs that a portion is taken to be measured when uneven sample analysis, for animal, then it is dirty to can use all tissues
Device is homogenized, and physiological saline must be used to clean the substances such as the blood of wherein surface mount before homogenization, so
It is mixed afterwards with certain proportion with physiological saline.Biomaterial nonuniform sample is generally semisolid or sticky sample, semisolid sample
This can set in refiner after mixing with physiological saline and wear into homogenate, and a small amount of methanol can be added in excrement or other liquid stir evenly, sticky
Sample can homogenize processing with ultrasonic wave.Nonuniform sample needs certain representativeness when acquiring, if taking whole tissues, needs
See clearly Chu position.It is all homogenized after weighing, part of it is taken to be measured.
The present invention disclose in cardiac muscular tissue's sample for addressing, be often referred to a kind of musculature of cardiac muscle cell's composition, broad sense
Cardiac muscle cell include composition sinoatrial node, room internal beam, atrioventricular junction portion, atrioventircular bundle (i.e. atrioventricular band) and Purkinje fiber etc. spy
The cardiac muscle cell and general heart muscle that have very broken up and ventricular muscles working cardial cell.Hippocampal tissue or hippocampus
It (Hippocampus) also known as hippocampal gyrus, hippocampus, is the title at a position in brain temporal lobe.Cortex is often referred to plant
One layer of parenchymal tissue between stem and root mesocuticle and vascular bundle, people or bio-tissue surface, for example, kidney cortex, plant
The cortex of object stem, may also mean that corticocerebral abbreviation, and the present invention discloses the middle typical cerebral cortex of selection and investigates.
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 is first
It selects between 3-5.5, preferably more preferred pH is more highly preferred to pH between 4.0-4.8 between 3.3-5.1.Meanwhile containing ammonia
The extracting solution of base acid is as sample to be tested, and when carrying out chromatograph mass spectrum analysis, extracting solution can flow through chromatographic column and mass spectral analysis ionization
Room, so the composition meeting testing result of extracting solution generates direct influence, the present invention discloses formic acid/ammonium formate buffering of selection
The requirement that composition in combined gas chromatography mass spectrometry analysis is easy gasification can be satisfied in liquid, acetic 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 disclose using containing formic acid/
For the acetonitrile solution of ammonium formate as chromatography mobile phase, mobile phase identical with extracting solution constituent can be further
Promote the separation and detection of amino acid in biological organization sample.
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.
During the present invention discloses, as the methanol solution of biological organization sample processing, concentration can choose 20-50%, compared with
For preferred 20-30%.The selection of methanol solution quantity determines according to sample properties and quantity, such as can choose 5-10 times of sample
The methanol solution of this volume.
In the preparation or method of completing the square of extracting solution disclosed by the invention, illustratively describe with formic acid and ammonium formate as buffering
The specific implementation of liquid component meets the buffer of open request of the present invention as other, such as acetic acid and ammonium acetate buffer body
System 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%.
The quantity of extracting solution also has an impact the extraction of amino acid in biological organization sample.During the present invention discloses, preferentially
The volume of selective extraction liquid is 250 times of biological tissue's homogenate or more, can effectively be extracted in biological tissue within this range
Amino acid composition, overcome the matrix effect of biological sample, and can satisfy extract after the separation of amino acid and detection are needed
It asks.
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；
Amino by the Performance discovery to above four kinds of extracting solutions, after the processing of the above extracting solution, in sample
Acid can be extracted efficiently, peak shape and respective strengths different from, for example, result is better than in extracting solution (2) and (4)
(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.
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, mobile phase
Organic phase be acetonitrile, wherein contain 1mmolL-1(mol·L-1It can be expressed as M, mmolL-1Can be expressed as mM) formic acid
Ammonium and 0.2% formic acid, the water phase of mobile phase is ultrapure water, wherein containing 1mmolL-1Ammonium formate and 0.1% formic acid.
During the present invention discloses, internal standard uses deuterated phenylalanine standard items, and process for preparation may include taking deuterated benzene third
Propylhomoserin standard items, accurately weighed, being configured to concentration with ultrapure water is 1mgmL-1Internal standard mother liquor, then as needed with extract
Liquid 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, prepare
At 10 μ gmL-1Inner mark solution, can be by 10 μ L, 10 μ gmL-1Inner mark solution is mixed with 990 μ L extracting solutions, is vortexed mixed
It is even, it is configured to 100ngmL-1Inner mark solution.
During the present invention discloses, the extracting solution of use and the mobile phase of liquid chromatogram can have identical constituent component, this
The extraction of amino acid in biological organization sample is not only contributed to, and is conducive to efficiently separating in analysis phase different aminoacids
It is detected with complete, moreover, can choose the extracting solution and stream that constituent component is identical, content is close as more preferred scheme
Dynamic phase, it is more preferred to, the component and concentration of extracting solution and the concentration of component of liquid chromatogram initial liquid phase completely corresponding one
When cause, has and more preferably analyze 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 biological organization sample processing method is related to, and
There is no its processing sequence of considered critical, if not otherwise specified, or within the scope of testing allows, can 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 being convenient for sample
Present treatment or other purposes, the step of needs, such as the packing of sample, the preparation or preformulation of extracting solution, the increasing of these steps
It adds deduct few, 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 without detection method certainty logical requirements, can according to actually detected demand,
It makes the appropriate adjustments, has no effect on testing result, and can according to need and increase other steps, such as increase repeatability and investigate step
Suddenly, the rate of recovery investigates step, also can according to need and reduces some steps, as amino acid standard curve (or linear investigates step
Suddenly) or mother liquor step etc., as long as the change that the needs of meeting amino acid detection is done, does not affect disclosed by the invention
Spirit Essence.
The present invention discloses being combined for liquid chromatography mass for offer and directly detects amino in biological tissue's nonuniform sample
The reagent packet of acid, can be the form of reagent packet, is also possible to kit or other facilitate the form of offer；For biological sample
The reagent of extracting solution is handled, offer form does not change its function used as extracting solution, so will not change it
Substantive content, the variation of any other form, all within spirit disclosed by the invention.
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 cardiac muscular tissue's 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), 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: Milli-
The ultrapure water purification system preparation of Q system.
3. sample
Cardiac muscular tissue's sample comes from Wistar rat, specification, the reasonable operation stream according to biological tissue's nonuniform sample
Journey 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, precise weighing, to homocysteine, L-2 aminoadipic acid and tyrosine,
It is formulated as 100 μ gmL respectively with the aqueous solution containing 0.1% formic acid-1、1mg·mL-1、250μg·mL-1Standard items mother liquor, it is right
Remaining amino acid, adding ultrapure water to be configured to concentration respectively is 1mgmL-1Standard items mother liquor；As needed with extracting solution to matching
The standard items mother liquor of system is diluted, and prepares hybrid standard product, respectively using S/N (the ratio between signal and noise) be greater than 3,10 as
Detection limit (LOD) and quantitative limit (LOQ), LOD and LOQ concentration are shown in Table 4, in hybrid standard product spectrogram such as Fig. 1 rat heart muscle tissue
29 kinds of amino acid standard LC-MS/MS spectrums.
7. extracting solution is prepared
Organic phase: the ammonium formate mixing of 85mL acetonitrile, 85uL 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
Cardiac muscular tissue is cleaned with physiological saline, is dried with dust-free paper, shreds and mixes, precision weighs 0.4994g, adds 8
20% methanol of amount homogenate again, is distributed into L/ parts of 4 μ, -80 DEG C of refrigerator freezings.The homogenate dispensed is taken out, 991 μ L are added and mention
Liquid is taken, extracting solution is 85% acetonitrile solution, wherein being added in 5 μ L comprising 0.1% formic acid and the ammonium formate of 1.7mmol/L
Mark, is inside designated as the deuterated phenylalanine of 100ng/mL, 1min, 12000rpm, 4 DEG C of centrifugation 5min of vortex mixed take supernatant to wait for
It surveys.Testing result is shown in 29 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 2 rat heart muscle tissue samples.
9. pattern detection
9.1 detection method linear relationships are investigated
3 cardiac muscular tissue's amino acid standard 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, as a result such as the following table 4, r value show that this method is linearly good between 0.9801-0.9999.
The linear equation of 29 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 4. rat heart muscle tissue 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, the RSD (%) of 29 kinds of amino acid peak areas as shown in table 6 below, between 1.05-12.84, show this method
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, the RSD (%) of 29 kinds of amino acid peak areas as shown in table 5 below, between 1.45-30.84, show 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 table 5 below, the RSD (%) of 29 kinds of amino acid peak areas shows this method between 1.15-14.21
Repeatability it is good.
The day to day precision of 29 kinds of amino acid, withinday precision and method repeatability in 5. rat heart muscle tissue 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: rate of recovery %=(measured 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
Between 70.76%-124.35%, show that this method is accurate and reliable.
The rate of recovery of 29 kinds of amino acid in 6. cardiac muscular tissue of table
9.4 freeze-thaw stabilities are investigated
12 parts of sample dispensed are taken, are divided into 3 groups, every group of 4 samples are distinguished freeze thawing 3 times, each 12h, by sample process
Method prepares sample to be tested solution, is measured, and the results are shown in Table 7, the RSD (%) of peak area shows between 1.03-18.53
Stablize by 3 freeze thawing samples.
The freeze-thaw stability of 7. 29 kinds of amino acid of cardiac muscular tissue of table
The detection of cortical tissue's sample.
1. laboratory apparatus
With embodiment 1.
2. reagent
Standard items: glycine (Glycine), Beta-alanine (β-Alanine), alanine (Alanine), gamma-amino fourth
Acid (γ-Aminobutyric acid), serine (Serine), histamine (Histamine), proline (Proline), figured silk fabrics ammonia
Acid (Valine), threonine (Threonine), cysteine (Cysteine), taurine (Taurine), leucine
(Leucine), trans--hydroxyproline (trans-4-Hydroxy-L-Proline), asparagine (Asparaginate), bird
Propylhomoserin (Ornithine), homocysteine (Homocysteine), lysine (Lysine), glutamic acid (Glutamic
Acid), methionine (Methionine), phenylalanine (Phenylalanine), 1- methyl-histidine (1-Methyl-
Histidine), citrulling (Citrulline), tyrosine (Tyrosine) tryptophan (Tryptophan), carnosine
(Carnosine), folic acid (Folic acid) is purchased from sigma company, the U.S., glutamine (Glutamine), histidine
(Histidine) it is 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
Brain cortical tissue's sample come from Sprague-Dawley rat, according to biological tissue's nonuniform sample specification,
Reasonable operating process 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 amino acid standard appropriate, it is accurately weighed；To homocysteine, tyrosine, folic acid, with containing 0.1% formic acid
Aqueous solution be formulated as 100 μ gmL respectively-1、250μg·mL-1、100μg·mL-1Standard items mother liquor, to remaining amino acid,
Adding ultrapure water to be configured to concentration respectively is 1mgmL-1Standard items mother liquor；It is diluted, and prepared with extracting solution as needed
Hybrid standard product are greater than 3,10 as detection limit (LOD) and quantitative limit (LOQ), LOD and LOQ concentration using S/N respectively and are shown in Table 8,
28 kinds of amino acid LC-MS/MS spectrums (standard items) in hybrid standard product spectrogram such as Fig. 3 rat heart muscle tissue samples.
7. extracting solution is prepared
With embodiment 1.
8. sample preparation
Brain cortical tissue is cleaned with physiological saline, dust-free paper is dried, precise weighing 0.7545g, adds 5 times of 20% first of amount
Alcohol homogenate, is distributed into L/ parts of 4 μ, -80 DEG C of refrigerator freezings.The homogenate dispensed is taken out, 991 μ L extracting solutions, extracting solution is added
For 85% acetonitrile solution, it includes the ammonium formates of 0.1% formic acid and 1.7mmol/L, and 5 μ L internal standards are added, are inside designated as
The deuterated phenylalanine of 100ng/mL, vortex mixed 1min, 12000rpm, 4 DEG C of centrifugation 5min, takes supernatant, to be measured.
Testing result is shown in 28 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 4 rat heart muscle tissue samples.
9. pattern detection
9.1 linear relationships are investigated
9 cortical tissue's amino acid standard of table
The preparation of linear mother liquor.Cortical tissue's 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 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) is linearly returned with concentration (pg/ μ L)
Return, calculate r, as a result such as the following table 8, r value show that this method is linearly good in 0.9746-0.9998.
The linear equation of 28 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 8. brain cortical tissue 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
It is fixed, the RSD (%) of 28 kinds of amino acid peak areas as shown in the following table 10, in the day between 0.81-34.59, showing 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 28 kinds of amino acid peak areas as shown in the following table 10, between 0.33-29.89, shows that this method is smart in the daytime
Density 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 28 kinds of amino acid peak areas shows we between 3.32-20.32
The repeatability of method is good.
The day to day precision of 28 kinds of amino acid, withinday precision and method repeatability in 10. brain cortical tissue 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-background)/additional amount × 100%, as shown in table 11 below, the rate of recovery is in 74.50- by the RSD (%) of the rate of recovery and its peak area
129.76%, show that this method is accurate and reliable.
The rate of recovery of 28 kinds of amino acid in 11. cerebral cortex 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, the RSD (%) of peak area 4.27-21.74 it
Between, show that, by 3 freeze thawing, sample is stablized.
The freeze-thaw stability of 12. 28 kinds of amino acid of brain cortical tissue of table
Hippocampal tissue pattern detection
1. laboratory apparatus
With embodiment 1.
2. reagent
Standard items: glycine (Glycine), Beta-alanine (β-Alanine), alanine (Alanine), serine
(Serine), histamine (Histamine), proline (Proline), valine (Valine), threonine (Threonine), half
Cystine (Cysteine), trans--hydroxyproline (trans-4-Hydroxy-L-Proline), asparagine
(Asparaginate), homocysteine (Homocysteine), glutamic acid (Glutamic Acid), methionine
(Methionine), L-2 aminoadipic acid (L-2-Aminoadipic acid), phenylalanine (Phenylalanine), smart ammonia
Sour (Arginine), citrulling (Citrulline), carnosine (Carnosine) are purchased from sigma company, the U.S., glutamine
(Glutamine) it is 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:
The ultrapure water of the ultrapure water purification system preparation of Milli-Q system.
3. sample
Hippocampal tissue sample comes from Wistar rat, according to non-homogeneous biological sample requirement of experiment, according to specification, reasonably
Operating process 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
Appropriate standard items are taken, it is accurately weighed；To homocysteine, L-2 aminoadipic acid, with the water for containing 0.1% formic acid
Solution is formulated as 100 μ gmL respectively-1、1mg·mL-1Standard items mother liquor ultrapure moisture is added to remaining amino acid standard
Not being configured to concentration is 1mgmL-1Standard items mother liquor, be diluted as needed with extracting solution, and prepare hybrid standard product,
It is greater than 3,10 as detection limit (LOD) and quantitative limit (LOQ) using S/N respectively, LOD and LOQ concentration is shown in Table 14, hybrid standard product spectrum
Figure such as 20 kinds of amino acid LC-MS/MS spectrums (standard items) in Fig. 5 Rat hippocampus.
7. extracting solution is prepared
With embodiment 1.
8. sample preparation
Hippocampal tissue is cleaned with physiological saline, dust-free paper is dried, and is shredded and is mixed, and precision weighs 0.0831g, adds 5 times
The homogenate of 20% methanol is measured, L/ parts of 4 μ, -80 DEG C of refrigerator freezings are distributed into.The homogenate 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 supernatant
Liquid is to be measured.
Testing result is shown in 20 kinds of amino acid LC-MS/MS spectrums (sample) in Fig. 6 Rat hippocampus.
9. pattern detection
9.1 linear relationships are investigated
13 hippocampal tissue amino acid standard of table
The preparation of linear mother liquor.Extracting solution and 13 amino acid standard of table is taken to be formulated as 2mL mother liquor, vortex 1min (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 0.5ng/mL
Deuterated phenylalanine calibrated as internal standard compound, by set chromatographic condition and Mass Spectrometry Conditions, sample introduction is analyzed, with each amino acid
Peak area ratio with internal standard compound is ordinate (Y), is that abscissa (X) carries out linear regression with concentration (pg/ μ L), calculates r, knot
The linear equation of 20 kinds of amino acid, the range of linearity and detection limit and quantitative limit, r value exist in 14 Rat hippocampus of fruit such as table
Between 0.9901-0.9999, show that this method is linearly good.
The linear equation of 20 kinds of amino acid, the range of linearity and detection limit and quantitative limit in 14. Rat hippocampus 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 20 kinds of amino acid peak areas as shown in table 15 below, between 0.76-28.14, shows the in a few days smart of this method
Density 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 20 kinds of amino acid peak areas as shown in table 15 below, between 0.37-28.64, shows that this method is accurate in the daytime
Degree 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 15 below, the RSD (%) of 20 kinds of amino acid peak areas shows this method between 2.32-6.88
Repeatability it is good.
The day to day precision of 20 kinds of amino acid, withinday precision and method repeatability in 15 Rat hippocampus 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
Between 71.45%-128.76%, show that this method is accurate and reliable.
The rate of recovery of 20 kinds of amino acid in 16. Rat hippocampus 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 is 4, freeze thawing 3 times respectively, each 12h, by preceding sample
Processing method prepares sample to be tested solution, is measured, and the results are shown in Table 17, the RSD (%) of peak area 1.41-18.06 it
Between, show that, by 3 freeze thawing, sample is stablized.
The freeze-thaw stability of 20 kinds of amino acid in 17. Rat hippocampus of table
Claims (4)
1. the method that liquid chromatography mass combination directly detects amino acid in biological tissue's nonuniform sample, which is characterized in that packet
It includes:
Biological tissue's nonuniform sample is handled with extracting solution；
Amino acid standard is prepared；
Biological tissue's nonuniform sample includes cardiac muscular tissue, brain cortical tissue and hippocampal tissue；
Wherein, extracting solution processing biological tissue's nonuniform sample specifically includes:
(i) biological tissue is cleaned with physiological saline, is dried, shredded and mixed；
(ii) it weighs, adds 20-30% methanol solution to be homogenized, obtain homogenate；
(iii) homogenate is put into -80 DEG C of refrigerator freezings；
(iv) by the homogenate 4-6 μ L of freezing, it is greater than 250:1 according to the volume ratio of extracting solution and homogenate, is mixed with extracting solution；
(v) the deuterated phenylalanine of internal standard, vortex mixed, body of the deuterated phenylalanine of internal standard in final sample to be tested solution is added
Product content is 0.5%；
(vi) 12000rpm, 4 DEG C of centrifugations, take supernatant, to be measured；
The amino acid standard is prepared
(a) cardiac muscular tissue's amino acid detects
To homocysteine, L-2- aminoadipic acid, tyrosine, it is formulated as 100 μ g respectively with the aqueous solution containing 0.1% formic acid
mL-1、1mg·mL-1、250μg·mL-1Standard items mother liquor；
To remaining amino acid, the standard items mother liquor of 1mg/mL is configured to ultrapure water；
With extracting solution dilution standard product mother liquor, it is configured to hybrid standard product；
(b) cortical tissue's amino acid detects
To homocysteine, tyrosine, folic acid, it is formulated as 100 μ gmL respectively with the aqueous solution containing 0.1% formic acid-1、250μ
g·mL-1、100μg·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；
(c) hippocampal tissue amino acid detects
To homocysteine, 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 standard, 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 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；
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.
4. one kind directly detects biological tissue's nonuniform sample for any one of the claim 1-3 liquid chromatography mass combination
The reagent packet of the method for middle amino acid, including following component:
Acetonitrile, concentration 85%；
Buffer, the ammonium formate that the formic acid and concentration that the buffer is 0.1% by concentration are 1.7mM form；
The pH of the extracting solution is 4.32.
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| 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 |
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