CN109633030A - A kind of method that ultra performance liquid chromatography-QQ-TOF mass spectrometry detects amino acid in animal body fluid or tissue samples - Google Patents
A kind of method that ultra performance liquid chromatography-QQ-TOF mass spectrometry detects amino acid in animal body fluid or tissue samples Download PDFInfo
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Abstract
The invention discloses the methods of amino acid in a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection animal body fluid or tissue samples, specifically: after extracting processing to animal body fluid or tissue samples, sample carries out qualitative and quantitative analysis to amino acid therein by ultra performance liquid chromatography-QQ-TOF mass spectrometry detection.The method of amino acid can synchronize detection animal body fluid and organize 26 kinds of amino acid in equal samples in a kind of ultra performance liquid chromatography that the present invention establishes-QQ-TOF mass spectrometry detection animal body fluid or tissue samples.It is carried out using multiple-reaction monitoring mass spectrum qualitative and quantitative, the instrument detection time of each sample only needs 25 minutes, is 25% or so of existing method time, and pre-treating method is simple, without derivative, each amino acid can be transferred through extracting selection ion and being separated well.
Description
Technical field
The invention belongs to animal foodstuff detection technique field, it is related to amino acid in a kind of detection animal body fluid or tissue samples
Method, in particular to amino in a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection animal body fluid or tissue samples
The method of acid.
Background technique
Amino acid is both that the substrate of synthetic proteins matter can be used as letter again as nitrogen main source needed for growth of animal
Number molecule plays the role of vital in growth of animal, muscle protein deposition etc..Therefore, amino acid is in animal blood
Slurry, organizes the accurate quantitative analysis measurement in equal samples for evaluating the nutritional need level of animal, finding amino acid in animal at muscle
Intracorporal synthesis provides strong technical support with the metabolic rule etc. decomposed.
For most of amino acid and the like, hydrophily is not easy by force to retain on reversed-phase column very much, soon
It is rinsed out, and since without strong ultraviolet absorption groups such as phenyl ring, the ultra-violet absorption spectrum of entire molecule is very weak, mesh
Before, the method for free amino acid mainly uses the liquid phase of column front derivation or post-column derivation in quantitative detection muscle, blood equal samples
Chromatography, common column front derivation agent mainly have phenyl isothiocyanate (PITC), phthalic aldehyde (OPA), 6- aminoquinoline-N-
Hydroxysuccinimidyl acylimino carbamate (AQC), 9- fluorene methyl methylchloroformate (FMOC) and 2,4-dinitrofluorobenzene
(DNFB) etc., post-column derivation agent is usually ninhydrin, and the defect of liquid-phase chromatography method essentially consists in the effect of the separation to complex matrices
Fruit is bad, since blood sample, muscle equal samples are a complicated matrix, there is many kin small molecule compounds, meeting
Interference is generated to the chromatographic isolation peak of target compound, especially for low-abundance amino acid, be often difficult it is accurate qualitative and
It is quantitative, cause resultant error very big, while the common liquid chromatography of either column front derivation or post-column derivation, in instrument
The time of upper analysis is all very long, and a sample usually requires or so 2 hours of operation.And for PITC derivative reagent, to color
Spectrum column has very strong destruction, and the derivative products after OPA is derivative are very unstable, and AQC purchase is again expensive, and ninhydrin is again
A kind of very strong organic compound of carcinogenicity.Therefore the defect of existing detection technique is considered, it would be highly desirable to which developing one kind can be quasi-
Really quantitative determine the new detection technique of a variety of free amino acids.
Liquid chromatography-mass spectrometry (LC-MS), using liquid chromatogram as separation system, mass spectrum is detection system for it.
Sample is separated in mass spectrum part and mobile phase, after being ionized, through mass spectrographic mass analyzer by fragment ion by mass number point
It opens, obtains mass spectrogram through detector.LC-MS embodies the complementation of chromatography and mass spectrum advantage, by chromatography to the height of complex sample
Separating capacity has the advantages that highly selective, highly sensitive and is capable of providing relative molecular mass in conjunction with structural information with MS
Get up, be widely used in detection and analysis field, is commonly used to detect difficult volatile materials.Not yet discovery utilizes at present
LC-MS measures the research report of amino acid in animal body fluid or tissue samples, and currently associated research still belongs to blank.
Summary of the invention
For needing column front derivation existing for existing detection technique or post-column derivation, detection time be too long, detection error is big
Defect, the purpose of the present invention is to provide a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection animal body fluid or groups
The method for knitting amino acid in sample, pre-treatment is simple, without deriving, greatly shortens detection time, greatly improves the choosing of detection
Selecting property and sensitivity.
The present invention is achieved by the following technical solutions:
A kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detects the side of amino acid in animal body fluid or tissue samples
Method, comprising the following steps:
After extracting processing to animal body fluid or tissue samples, sample passes through ultra performance liquid chromatography-series connection level four bars
Mass Spectrometer Method carries out qualitative and quantitative analysis to amino acid therein；
Wherein, the animal body fluid or tissue samples are animal blood plasma, animal muscle or animal's liver；
The amino acid is alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, different bright ammonia
Acid, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ox
Sulfonic acid, hydroxyproline, asparagine, glutamine, citrulling, aminobutyric acid, beta-aminobutyric acid, 26 kinds of amino acid of ornithine.
Further improvement of the present invention scheme are as follows:
The chromatographic condition of the ultra performance liquid chromatography are as follows:
Chromatographic column: WatersAtlantis T3 chromatographic column 4.6 × 250mm, 5 μm；
Column temperature: 30 DEG C；
Sample volume: 2ul；
Mobile phase: A:20mM ammonium acetate solution, with chromatographically pure acetic acid tune pH to 5.0；B:80% acetonitrile solution；
Flow velocity: 0.80mL/min；
Type of elution: gradient elution.
The gradient elution program are as follows:
。
The QQ-TOF mass spectrometry testing conditions are as follows:
Ion source: ESI；Detection mode, positive ion mode, more reaction detections；
Atomization gas temperature: 350 DEG C, atomization gas air-flow: 5Lmin-1；
Sheath temperature degree: 400 DEG C, sheath gas air-flow: 12Lmin-1；
Capillary voltage: 3500V；
Orifice potential: 1500V；
Collision gas flow velocity: 6Lmin-1。
Further improvement project of the invention are as follows:
The processing method of the animal blood plasma are as follows:
It takes animal blood plasma that extracting solution is added, is vortexed after concussion, sample is centrifuged 10min at 4 DEG C with 14500rpm；It collects
Supernatant is simultaneously spin-dried for；Solvent is added to residue to redissolve, be vortexed and 10min is centrifuged with 14500rpm at 4 DEG C again；By supernatant
Liquid is transferred to sample introduction bottle for ultra performance liquid chromatography-QQ-TOF mass spectrometry detection.
Wherein, the volume ratio of the blood plasma, extracting solution and solvent is 1:4:1.The extracting solution is the first that volume ratio is 1:1
The mixing of alcohol and acetonitrile；The solvent is the mixing of methanol and water that volume ratio is 1:1.
The processing method of the animal muscle or animal's liver are as follows:
It takes animal muscle or liver specimens to grind in liquid nitrogen, mixed extract is added, be vortexed after concussion, by sample dry
4h is stood on ice, then 10min is centrifuged with 14500rpm at 4 DEG C；It collects supernatant and methanol extract liquid, whirlpool is added into residue
It revolves oscillation 1min to be centrifuged sample again after standing 30min on dry ice, takes supernatant and extract supernatant with first time and mix
It closes, supernatant vacuum is spin-dried for；Sample is redissolved with solvent, be vortexed and 10min is centrifuged with 14500rpm at 4 DEG C again.It will be upper
Clear liquid is transferred to sample introduction bottle for ultra performance liquid chromatography-QQ-TOF mass spectrometry detection.
Wherein, the mass volume ratio of the animal muscle or liver, mixed extract, methanol extract liquid and solvent is 1mg:
33.3μL:26.7μL:6.7μL.The mixed extract is the mixing of methanol and water that volume ratio is 8:2；The solvent is body
Mixing of the product than the methanol and water that are 1:1.
The invention has the benefit that
In a kind of ultra performance liquid chromatography that the present invention establishes-QQ-TOF mass spectrometry detection animal body fluid or tissue samples
The method of amino acid can synchronize detection animal body fluid and organize 26 kinds of amino acid in equal samples.Using multiple-reaction monitoring mass spectrum
It carries out qualitative and quantifies, it is 25% or so of existing method time, and preceding that the instrument detection time of each sample, which only needs 25 minutes,
Processing method is simple, and without deriving, each amino acid can be transferred through extracting selection ion and being separated well.
The present invention carries out firsts and seconds scanning using mass spectrum, obtains the mass spectrum letter of its parent ion and fragment fragment ion
Breath.It is quantified with the peak area of one of them correspondingly high fragment daughter ion, 2 fragment ions progress are qualitative, are greatly improved
The selectivity and sensitivity of detection, sensitivity at least can be improved 10 times or more, reaches ppb or ppt grades.
The detection limit range of method amino acid standard specimen of the invention be 0.001ng/mL-0.5ng/mL, linearly dependent coefficient >
0.99, accurate mother ion mass-to-charge ratio relative deviation is less than 3ppm.
Detailed description of the invention
26 kinds of amino acid that Fig. 1 is 10 μM mix the total ion chromatogram of standard specimen；
The multiple-reaction monitoring of glycine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 2 is 10 μM；
The multiple-reaction monitoring of alanine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 3 is 10 μM；
The multiple-reaction monitoring of serine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 4 is 10 μM；
The multiple-reaction monitoring of proline extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 5 is 10 μM；
The multiple-reaction monitoring of valine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 6 is 10 μM；
The multiple-reaction monitoring of threonine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 7 is 10 μM；
The multiple-reaction monitoring of taurine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 8 is 10 μM；
The multiple-reaction monitoring of hydroxyproline extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Fig. 9 is 10 μM；
The multiple-reaction monitoring of isoleucine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 10 is 10 μM；
The multiple-reaction monitoring of leucine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 11 is 10 μM；
The multiple-reaction monitoring of asparagine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 12 is 10 μM；
The multiple-reaction monitoring of aspartic acid extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 13 is 10 μM；
The multiple-reaction monitoring of lysine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 14 is 10 μM；
The multiple-reaction monitoring of the mixed standard specimen Glutamic Acid for 26 kinds of amino acid that Figure 15 is 10 μM extracts chromatography of ions figure；
The multiple-reaction monitoring of methionine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 16 is 10 μM；
The multiple-reaction monitoring of histidine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 17 is 10 μM；
The multiple-reaction monitoring of phenylalanine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 18 is 10 μM；
Arginic multiple-reaction monitoring extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 19 is 10 μM；
The multiple-reaction monitoring of tyrosine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 20 is 10 μM；
The multiple-reaction monitoring of tryptophan extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 21 is 10 μM；
The multiple-reaction monitoring of cystine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 22 is 10 μM；
The multiple-reaction monitoring of ornithine extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 23 is 10 μM；
The multiple-reaction monitoring of citrulling extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 24 is 10 μM；
The multiple-reaction monitoring of the mixed standard specimen glutamine for 26 kinds of amino acid that Figure 25 is 10 μM extracts chromatography of ions figure；
The multiple-reaction monitoring of aminobutyric acid extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 26 is 10 μM；
The multiple-reaction monitoring of beta-aminobutyric acid extracts chromatography of ions figure in the mixed standard specimen for 26 kinds of amino acid that Figure 27 is 10 μM；
Figure 28 is the total ion chromatography spectrum of amino acid content in animal blood plasma；
Figure 29 is amino acid total ion chromatogram spectrum in animal muscle；
Figure 30 is amino acid total ion chromatogram spectrum in animal's liver.
Specific embodiment
1, instrument and reagent
High performance liquid chromatography-tandem mass: 1200Series HPLC--6460Triple Quad MS, U.S. Agilent
Technologies company's T hermo Fisher company；Vacuum rotating concentrating instrument: Concentratorplus type, Eppendorf
Company, Germany；Centrifuge: Beckman company, the U.S.；Ultrapure water instrument: MilH-QBiocel type, Millipore company, the U.S.；
Turbine mixer: HQ-60-II, Beijing Tongfang company, China；Micropipettor: P-5000, P-200, P-100, P-20 and P-2
Type, Eppendorf company, Germany.
HPLC grades of acetonitriles, methanol are purchased from Sigma Co., USA；(99.5%HPLC grades) of ammonium acetate are purchased from Beijing enlightening section equine
Skill Co., Ltd；Water (chromatographically pure), acetic acid (chromatographically pure) are purchased from U.S. Fisher company；26 kinds of amino acid (alanine, smart ammonia
Acid, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylpropyl alcohol ammonia
Acid, proline, serine, threonine, tryptophan, tyrosine, valine, taurine, hydroxyproline, asparagine, glutamy
Amine, citrulling, aminobutyric acid, beta-aminobutyric acid, ornithine) standard items are purchased from sigma company, the U.S..
2, testing conditions
The chromatographic condition of ultra performance liquid chromatography are as follows:
Chromatographic column: WatersAtlantis T3 chromatographic column 4.6 × 250mm, 5 μm；
Column temperature: 30 DEG C；
Sample volume: 2ul；
Mobile phase: A:20mM ammonium acetate solution, with chromatographically pure acetic acid tune pH to 5.0；B:80% acetonitrile solution；
Flow velocity: 0.80mL/min；
Type of elution: gradient elution.
The gradient elution program are as follows:
。
QQ-TOF mass spectrometry testing conditions are as follows:
Ion source: ESI；Detection mode, positive ion mode, more reaction detections；
Atomization gas temperature: 350 DEG C, atomization gas air-flow: 5Lmin-1；
Sheath temperature degree: 400 DEG C, sheath gas air-flow: 12Lmin-1；
Capillary voltage: 3500V；
Orifice potential: 1500V；
Collision gas flow velocity: 6Lmin-1。
The optimization of liquid-phase condition and Mass Spectrometry Conditions is carried out using high performance liquid chromatography-quadrupole rods tandem mass spectrometry, it is final to use
Waters Atlantis T3 chromatographic column (4.6 × 250mm, 5 μm) is separated, gradient elution.Mixed sample is on instrument
Detection time only needs 25 minutes, and 26 kinds of amino acid have all obtained good separation in chromatography.Using mass spectrum carry out level-one and
Second level scanning, obtains the Information in Mass Spectra of its parent ion and fragment fragment ion.With one of them correspondingly high fragment daughter ion
Peak area is quantified, and 2 fragment ions progress are qualitative, greatly improves the selectivity and sensitivity of detection, sensitivity is at least
It can be improved 10 times or more, reach ppb or ppt grades.
Qualitative determination: each tested 1 parent ion of component selection, 2 daughter ions, under same experimental conditions, in sample
Corresponding retention time deviation is within ± 2.5% in the retention time and hybrid standard product of test substance, and in sample spectrogram
The accurate mass-to-charge ratio of parent ion and daughter ion that each component measures surely and relative deviation < 3ppm of theoretical value, and ion is opposite
Abundance of ions is compared with the relative ion abundance of corresponding qualitative ion in the standard items working solution that concentration is close, and deviation is not
More than 30%, then can determine whether as there are corresponding determinands in sample.The accurate matter lotus of chemical formula, the parent ion of 26 kinds of amino acid
1 is shown in Table than, retention time and fragments characteristic.
The accurate mass-to-charge ratio of chemical formula, parent ion, retention time and the fragments characteristic of table 1,26 kind of amino acid
。
Quantitative determination: to mixed mark standard items working solution sample introduction, parent ion peak is scanned to be tested component level-one in standard solution
Area is ordinate, and being tested concentration of component is that abscissa draws standard curve, is quantified with standard curve to sample, sample is molten
The response of determinand should all be within the scope of the setting-out line that instrument detects in liquid.
3, standard curve
The configuration of singly mark solution: measuring (title) respectively and 26 kinds of amino acid standards taken to be placed in 26 10mL volumetric flasks, chromatography
Pure water constant volume, is made into the singly mark solution of concentration 100mM.
The configuration of mixed standard solution measures 26 kinds of amino acid standard solution 0.1mL in 1 10mL volumetric flask respectively
In, the water constant volume of chromatographically pure is made into the mixed standard solution that 26 kinds of amino acid concentrations are 1mM.
It makes standard curve: the mixed standard solution of configuration is drawn into 1mL, equimultiple dilutes step by step with chromatographic grade water, obtains
The mixed standard specimen of 26 kinds of amino acid of 10 various concentrations of 500~2.5 concentration, successively to the mixed standard specimen of each concentration according to upper
Ultra performance liquid chromatography on testing conditions-QQ-TOF mass spectrometry detection is stated, using amino acid peak area as ordinate Y, amino acid
Concentration is abscissa X, and drafting obtains 26 standard curves, and design parameter is shown in Table 2.
The standard curve relevant parameter of 2 26 kinds of amino acid of table
As seen from the above table: the animal body fluid or tissue samples realize separation in 25min, in the detection range of 5~50M
It is interior that there is good linear relationship, coefficient R2It is all larger than 0.99.
4, the measurement of sample
Embodiment 1: the measurement of amino acid in animal blood plasma
It takes the animal plasma sample that lot number is 2018091415 to be placed on from the taking-up of -80 DEG C of refrigerator to thaw on ice.Take 200
800 μ L extracting solutions (methanol: acetonitrile volume ratio 1:1) is added in μ L blood plasma.It is vortexed after concussion, by sample with 14500rpm at 4 DEG C
It is centrifuged 10min.Collect supernatant and in traditional vacuum thickener (Concentrator plus type, Eppendorf company, moral
State) in be spin-dried for.To residue be added 200 μ L volume ratio be 1:1 methanol and water redissolve, be vortexed and again at 4 DEG C with
14500rpm is centrifuged 10min.Supernatant is transferred to ultra performance liquid chromatography on sample introduction bottle-QQ-TOF mass spectrometry detection.
Testing conditions are as follows:
The chromatographic condition of ultra performance liquid chromatography are as follows:
Chromatographic column: Waters Atlantis T3 chromatographic column 4.6 × 250mm, 5 μm；
Column temperature: 30 DEG C；
Sample volume: 2ul；
Mobile phase: A:20mM ammonium acetate solution, with chromatographically pure acetic acid tune pH to 5.0；B:80% acetonitrile solution；
Flow velocity: 0.80mL/min；
Type of elution: gradient elution.
The gradient elution program are as follows:
。
QQ-TOF mass spectrometry testing conditions are as follows:
Ion source: ESI；Detection mode, positive ion mode, more reaction detections；
Atomization gas temperature: 350 DEG C, atomization gas air-flow: 5Lmin-1；
Sheath temperature degree: 400 DEG C, sheath gas air-flow: 12Lmin-1；
Capillary voltage: 3500V；
Orifice potential: 1500V；
Collision gas flow velocity: 6Lmin-1。
Amino acid content test map is as shown in figure 28 in animal blood plasma, the results are shown in Table 3.
Embodiment 2: the measurement of amino acid in animal muscle
Taking 15mg lot number is that 2018091515 animal muscle sample is ground in liquid nitrogen, and the extracting solution (first of 500 μ L is added
Alcohol: water volume ratio 8:2).It is vortexed after concussion, sample is stood into 4h on dry ice, 10min is centrifuged with 14500rpm at 4 DEG C.
It collects supernatant and 400 μ L methanol extract liquids, vortex oscillation 1min is added into residue.After standing 30min on dry ice, by sample
Product are centrifuged again, are taken supernatant and are mixed with first time extraction supernatant, supernatant vacuum is spin-dried for.It is with 100 μ L volume ratios
The methanol and water mixed solution of 1:1 redissolves sample, is vortexed and is centrifuged 10min at 4 DEG C again with 14500rpm.Supernatant is turned
Move to ultra performance liquid chromatography on sample introduction bottle-QQ-TOF mass spectrometry detection.
Testing conditions are with embodiment 1, and the content detection map of amino acid is as shown in figure 29 in animal muscle, the results are shown in Table 3.
Embodiment 3:: the measurement of amino acid in animal's liver
Taking 15mg lot number is that 2018101112 animal's liver sample is ground in liquid nitrogen, and the extracting solution (first of 500 μ L is added
Alcohol: water volume ratio 8:2).It is vortexed after concussion, sample is stood into 4h on dry ice, 10min is centrifuged with 14500rpm at 4 DEG C.
It collects supernatant and 400 μ L methanol extract liquids, vortex oscillation 1min is added into residue.After standing 30min on dry ice, by sample
Product are centrifuged again, are taken supernatant and are mixed with first time extraction supernatant, supernatant vacuum is spin-dried for.It is with 100 μ L volume ratios
The methanol and water mixed solution of 1:1 redissolves sample, is vortexed and is centrifuged 10min at 4 DEG C again with 14500rpm.Supernatant is turned
Move to ultra performance liquid chromatography on sample introduction bottle-QQ-TOF mass spectrometry detection.
Testing conditions are with embodiment 1, and the content detection map of amino acid is as shown in figure 30 in animal's liver, the results are shown in Table 3.
26 kinds of amino acid contents (n=3, means standard deviation) in the sample of 3 embodiment 1-3 of table
5, the verifying of method
The sensitivity of method is investigated by detection limit and quantitative limit.Wherein detection limit is defined as the 3 of noise signal intensity
Times, detection of 33 kinds of free amino acids in serum is limited to 0.003~0.75 μm of ol/L in this method, in muscle and liver specimens
In detection be limited to 0.003~0.31nmol/g.Quantitative limit is defined as 10 times of noise signal intensity, gained detection limit and quantitative
Limit is shown in Table 4.
The range of linearity of the 4 26 kinds of amino acid of table in LC-MS and the quantitative limit (n=3) in 3 kinds of different substrates
The accuracy and accuracy of method add recovery test by sample to investigate.Test result shows 26 kinds of amino acid
Accurate mother ion mass-to-charge ratio relative deviation be less than 3ppm, good linear relationship is presented within the scope of 2.5~250 μm of ol/L,
Related coefficient is all larger than 0.99, the quantitative limit in animal body fluid and muscle be respectively 0.01~0.24 μm of ol/L and 0.01~
0.90nmol/g (table 3), the rate of recovery is between 77.7%~124.8%, and relative standard deviation is less than 12.3% (table 5), explanation
Method is more reliable.
The rate of recovery and relative standard deviation (%) (n=5) of 26 kinds of amino acid in 5 matrix of table addition recycling sample
Note:1Number in bracket represents relative standard deviation.
Claims (10)
1. the method for amino acid in a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection animal body fluid or tissue samples,
Characterized by comprising the following steps:
After extracting processing to animal body fluid or tissue samples, sample passes through ultra performance liquid chromatography-QQ-TOF mass spectrometry
Detection carries out qualitative and quantitative analysis to amino acid therein；
Wherein, the animal body fluid or tissue samples are animal blood plasma, animal muscle or animal's liver；
The amino acid be alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine,
Leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ox sulphur
Acid, hydroxyproline, asparagine, glutamine, citrulling, aminobutyric acid, beta-aminobutyric acid, amino acid in ornithine 26.
2. a kind of ultra performance liquid chromatography according to claim 1-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the chromatographic condition of the ultra performance liquid chromatography are as follows:
Chromatographic column: Waters Atlantis T3 chromatographic column 4.6 × 250mm, 5 μm；
Column temperature: 30 DEG C；
Sample volume: 2 ul；
Mobile phase: A:20mM ammonium acetate solution, with chromatographically pure acetic acid tune pH to 5.0；B:80% acetonitrile solution；
Flow velocity: 0.80 mL/min；
Type of elution: gradient elution.
3. a kind of ultra performance liquid chromatography according to claim 2-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the gradient elution program are as follows:
4. a kind of ultra performance liquid chromatography according to claim 1-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the QQ-TOF mass spectrometry testing conditions are as follows:
Ion source: ESI；Detection mode, positive ion mode, more reaction detections；
Atomization gas temperature: 350 DEG C, atomization gas air-flow: 5L min-1；
Sheath temperature degree: 400 DEG C, sheath gas air-flow: 12 Lmin-1；
Capillary voltage: 3500 V；
Orifice potential: 1500 V；
Collision gas flow velocity: 6 Lmin-1。
5. a kind of ultra performance liquid chromatography according to claim 1-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the processing method of the animal blood plasma are as follows:
It takes animal blood plasma that extracting solution is added, is vortexed after concussion, sample is centrifuged 10 min at 4 DEG C with 14500 rpm；In collection
Clear liquid is simultaneously spin-dried for；Solvent is added to residue to redissolve, be vortexed and is centrifuged 10 min at 4 DEG C again with 14500 rpm；By supernatant
Liquid is transferred to sample introduction bottle for ultra performance liquid chromatography-QQ-TOF mass spectrometry detection.
6. a kind of ultra performance liquid chromatography according to claim 5-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the volume ratio of the blood plasma, extracting solution and solvent is 1:4:1.
7. a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection animal according to claim 5 or 6 any one
The method of amino acid in body fluid or tissue samples, it is characterised in that: the extracting solution is the methanol and acetonitrile that volume ratio is 1:1
Mixing；The solvent is the mixing of methanol and water that volume ratio is 1:1.
8. a kind of ultra performance liquid chromatography according to claim 1-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the processing method of the animal muscle or animal's liver are as follows:
It takes animal muscle or liver specimens to grind in liquid nitrogen, mixed extract is added, be vortexed after concussion, by sample on dry ice
4 h are stood, then are centrifuged 10 min at 4 DEG C with 14500 rpm；It collects supernatant and methanol extract liquid, whirlpool is added into residue
Rotation 1 min of oscillation, after 30 min are stood on dry ice, sample is centrifuged again, is taken supernatant and is extracted supernatant with first time
Mixing, supernatant vacuum is spin-dried for；Sample is redissolved with solvent, be vortexed and is centrifuged 10 min at 4 DEG C again with 14500 rpm,
Supernatant is transferred to sample introduction bottle and is used for ultra performance liquid chromatography-QQ-TOF mass spectrometry detection.
9. a kind of ultra performance liquid chromatography according to claim 8-QQ-TOF mass spectrometry detection animal body fluid or tissue
The method of amino acid in sample, it is characterised in that: the animal muscle or liver, mixed extract, methanol extract liquid and solvent
Mass volume ratio be 1mg:33.3 μ L:26.7 μ L:6.7 μ L.
10. a kind of ultra performance liquid chromatography-QQ-TOF mass spectrometry detection according to claim 8 or 9 any one is dynamic
The method of amino acid in object liquid or tissue samples, it is characterised in that: the mixed extract be volume ratio be 8:2 methanol with
The mixing of water；The solvent is the mixing of methanol and water that volume ratio is 1:1.
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| 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 |
| CN111638293A (en) * | 2019-08-30 | 2020-09-08 | 华南农业大学 | LC-MS/MS detection method for simultaneously determining amino acids and nucleotides in animal tissues |
| CN112051339A (en) * | 2020-07-09 | 2020-12-08 | 苏州旭辉检测有限公司 | Method for detecting aspartic acid in blood sample |
| CN112630338A (en) * | 2020-12-18 | 2021-04-09 | 中国科学院生态环境研究中心 | Detection method for detecting seven amino acids in earthworm body by reversed-phase high performance liquid chromatography tandem mass spectrometry |
| CN113030293A (en) * | 2021-02-05 | 2021-06-25 | 申友基因组研究院(南京)有限公司 | Tandem mass spectrometry method for rapidly detecting nine free amino acids in plasma |
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| 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 |
| CN112051339A (en) * | 2020-07-09 | 2020-12-08 | 苏州旭辉检测有限公司 | Method for detecting aspartic acid in blood sample |
| CN114624338A (en) * | 2020-12-10 | 2022-06-14 | 中国科学院深圳先进技术研究院 | Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry |
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| CN112630338A (en) * | 2020-12-18 | 2021-04-09 | 中国科学院生态环境研究中心 | Detection method for detecting seven amino acids in earthworm body by reversed-phase high performance liquid chromatography tandem mass spectrometry |
| CN112630338B (en) * | 2020-12-18 | 2023-02-24 | 中国科学院生态环境研究中心 | Detection method for detecting seven amino acids in earthworm body by reversed-phase high performance liquid chromatography tandem mass spectrometry |
| CN113030293B (en) * | 2021-02-05 | 2022-10-14 | 申友基因组研究院(南京)有限公司 | Tandem mass spectrometry for rapidly detecting nine free amino acids in blood plasma |
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| CN114236025B (en) * | 2021-12-09 | 2023-09-01 | 浙江博圣生物技术股份有限公司 | Method for determining 43 amino acids without ion pair reagent and non-derivatization |
| CN114509514A (en) * | 2022-01-13 | 2022-05-17 | 厦门医学院 | Method for detecting taurine and arginine in hippocampus trimaculatus by liquid chromatography-electrospray ionization tandem mass spectrometry |
| CN114441681A (en) * | 2022-01-28 | 2022-05-06 | 山东省食品药品检验研究院 | Method for measuring 20 free amino acids and L-hydroxyproline in special medical formula food |
| CN114441681B (en) * | 2022-01-28 | 2024-06-04 | 山东省食品药品检验研究院 | Method for determining 20 free amino acids and L-hydroxyproline in special medical formula food |
| CN115469043A (en) * | 2022-09-26 | 2022-12-13 | 中国农业科学院农业质量标准与检测技术研究所 | Liquid chromatography-tandem mass spectrometry detection method for free amino acids in ginger rhizome, overground stem and leaf |
| CN115480017A (en) * | 2022-10-12 | 2022-12-16 | 梧州市食品药品检验所 | Method for determining amino acid compounds in Liupu tea through high-resolution mass spectrometry |
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