CN108426961A - The method for detecting Pidolidone and L-Aspartic acid content in blood simultaneously - Google Patents
The method for detecting Pidolidone and L-Aspartic acid content in blood simultaneously Download PDFInfo
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- CN108426961A CN108426961A CN201810476876.1A CN201810476876A CN108426961A CN 108426961 A CN108426961 A CN 108426961A CN 201810476876 A CN201810476876 A CN 201810476876A CN 108426961 A CN108426961 A CN 108426961A
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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
- G01N30/02—Column chromatography
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Abstract
Method that is of the invention entitled while detecting L glutamic acid and L aspartate contents in blood, it demarcates standard solution using efficient liquid phase tandem mass spectrometer, and fitting, which obtains, respectively represents the calibration curve equation of L aminoglutaric acid concentrations as y1=a*x1+ the b and calibration curve equation y for representing L aspartic acid concentration2=c*x2+ d takes blood sample to be measured, to the blood sample to be detected after pre-treatment, is detected to sample to be measured using identical efficient liquid phase tandem mass spectrometer, obtains blood y to be measured1And y2Value, by the y of blood sample to be measured1And y2It substitutes into above-mentioned two calibration curve equation respectively, the relative concentration x of L glutamic acid and L aspartate contents and internal standard compound in blood sample to be measured is respectively obtained by calculating1And x2, internal standard compound concentration is known, and the concentration of L glutamic acid and L aspartate contents in blood sample to be detected can be obtained by calculating.
Description
Technical field
The present invention relates to amino acid detection techniques field, more particularly to a kind of detecting in blood Pidolidone and L- days simultaneously
The method of winter histidine content.
Background technology
Pidolidone (L-glutamic acid, L-Glu) is a kind of acidic amino acid, and molecule includes two carboxyls, chemistry
Entitled alpha-amido glutaric acid, molecular formula CsH9NO4, chemical structural formula is as shown in Figure 1.It is nitrogen metabolism in living organism
One of primary amino acid is nonessential amino acid, can be used as acidic amino acid and participates in metabolism；In addition, but also as excitability god
Information is participated in through mediator to transmit, and is related to proliferation, development, survival and the dead and learning and memory of neuron and spongiocyte
The Changes of Plasticity of closely related long term potentiation (LTP) and Long-term depression (LTD) cynapse transmission efficiency.
Under the pathologic conditions such as cerebral ischemia, while intracerebral L-Glu concentration increases, L-Glu concentration also accordingly increases in blood.Measure blood
Middle L-Glu elevated levels can reflect the variation of brain tissue ischemia degree.
L-Aspartic acid (L-aspartic acid, L-Asp), chemical name are L- (+)-aminosuccinic acid, for acid ammonia
One of base acid belongs to nonessential amino acid, molecular formula C4H7NO4, chemical structural formula is as shown in Figure 2.L-Asp is more in organism
The synthesis precursor of kind amino acid and purine, pyrimidine bases can be used as the carrier Cardiomyocytes conveying electrolyte of K+, Mg+ ion, to
Improve myocardium shrinkage function, while reducing oxygen consumption, in coronary artery circulation obstacle anoxic, has protective effect to cardiac muscle.And
Ornithine cycle is participated in, liver function, dispelling fatigue are enhanced.In addition, it is main emerging in mammalian central nervous system
It puts forth energy acidic amino acid class neurotransmitter, the concentration variation of specific region is closely related with Parkinson's disease in brain, shows as content
Significantly increase.The variation of Human Physiology situation can be reacted to a certain extent by measuring L-Asp levels in blood.
Currently, it is many in relation to the assay method of Pidolidone and L-Aspartic acid report in serum and blood plasma both at home and abroad,
In common method have：Automatic amino acid analyzer method, liquid chromatography, combined gas chromatography mass spectrometry, liquid chromatography mass
Combination method etc., in numerous methods, liquid chromatography or liquid phase tandem mass spectrometry are the prefered method for measuring vitamin.It is logical
Often, sample need to be quantitative with Instrumental Analysis again after derivatization method is handled, and there is complex pretreatment, disturbing factor is more, analyzes
The problems such as time is long, sensitivity is not high, qualitative, quantitative is not accurate enough.
Invention content
In view of the above technical problems, Pidolidone and L- asparagus fern ammonia in blood being detected simultaneously it is an object of the present invention to provide a kind of
Internal standard method is combined by the method for acid content with high performance liquid chromatography Mass Spectrometry, and this method is carried out in non-derivative method
Pre-treatment makes disturbing factor greatly reduce, and high specificity, high sensitivity, testing result are more accurate, while analysis time contracts
It is short, and by simplifying pre-treatment step, shorten sample processing time.
The technical solution adopted by the present invention is：
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：It includes
Following steps：
(1) calibration of standard solution A and standard solution B
Pipette 20 μ L at least three kinds of various concentration standard working solution A and 10 μ L mixing internal standard work respectively with pipettor first
Liquid is respectively placed in the standard solution A that at least three kinds various concentrations are mixed and made into centrifuge tube, and above-mentioned standard solution is being turned respectively
Speed is 100 μ L of addition protein precipitation reagent under 1500-2000rpm after vortex mixing 1-2min, and is 1500- in rotating speed
Under 2000rpm after vortex mixing 2-4min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, obtain supernatant
Liquid pipettes above-mentioned supernatant respectively, is detected to above-mentioned supernatant using efficient liquid phase tandem mass spectrometer, obtains at least three kinds
The chromatogram of the Pidolidone of various concentration and the standard solution A of internal standard compound, from the Pidolidone and internal standard of each above-mentioned concentration
Respectively obtain Pidolidone chromatographic peak area and internal standard compound chromatographic peak area in the chromatogram of the standard solution A of object, respectively more than
The Pidolidone chromatographic peak area of at least three various concentrations and the ratio of internal standard compound chromatographic peak area are stated as standard curve side
The ordinate y of journey1, in above-mentioned standard working solution A Pidolidone concentration and the internal standard compound concentration that mixes in internal standard working solution
Ratio be abscissa x as calibration curve equation1, the data of at least three kinds various concentrations of the above detection gained are carried out
Linear regression, it is y that fitting, which obtains calibration curve equation,1=a*x1+ b, and obtain weight coefficient a, b；
Pipette 20 μ L at least three kinds of various concentration standard working solution B and 10 μ L mixing internal standard working solutions point respectively with pipettor
It is not placed in centrifuge tube and is mixed and made into the standard solution B of at least three kinds various concentrations, be in rotating speed respectively by above-mentioned standard solution B
Under 1500-2000rpm after vortex mixing 1-2min, 100 μ L of protein precipitation reagent are added, and in the case where rotating speed is 1500-2000rpm
After vortex mixing 2-4min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, supernatant is obtained, is pipetted respectively
Above-mentioned supernatant is detected above-mentioned supernatant using efficient liquid phase tandem mass spectrometer, obtains at least three kinds of various concentrations
The chromatogram of the standard working solution B of L-Aspartic acid and internal standard compound, from the L-Aspartic acid of each above-mentioned concentration and internal standard compound
L-Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area are respectively obtained in the chromatogram of standard working solution B, with it is above-mentioned extremely
The chromatographic peak area of the L-Aspartic acid of few three kinds of various concentrations is with the ratio of internal standard compound chromatographic peak area as standard curve side
The ordinate y of journey2；With in above-mentioned standard working solution B L-Aspartic acid concentration and the internal standard compound mixed in internal standard working solution it is dense
Abscissa x of the ratio of degree as calibration curve equation2, the data of at least three kinds various concentrations of the above detection gained are carried out
Linear regression, it is y that fitting, which obtains calibration curve equation,2=c*x2+ d, and obtain weight coefficient c, d；
Criteria discussed above working solution A is the solution containing Pidolidone, and standard working solution B is to contain L-Aspartic acid
Solution, mixing internal standard working solution is molten containing Pidolidone and L-Aspartic acid mixing Isotopic Internal Standard standard items NSK-A
Liquid；
(a) preparation of standard reserving solution A and standard reserving solution B：
Accurately weighing Pidolidone standard items 6.77mg is placed in 10mL volumetric flasks, is dissolved with 50% methanol aqueous solution,
And constant volume obtains standard reserving solution A, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 month；
Accurately weighing L-Aspartic acid standard items 4.96mg is placed in 10mL volumetric flasks, is carried out with 50% methanol aqueous solution molten
Solution, and constant volume obtains standard reserving solution B, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 month；
(b) preparation of standard working solution A and standard working solution B：
Take appropriate step (a) Plays storing solution A, be diluted with the methanol aqueous solution of 40-60%, obtain containing 5 μM-
The Pidolidone standard working solution A of 1280 μM of concentration, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
Appropriate step (a) Plays storing solution B is taken, is diluted, is contained with the methanol aqueous solution of 40-60%
The L-Aspartic acid standard working solution B of 0.15625 μM of -160.00M μ concentration, and preserved under the conditions of -80 DEG C, the term of validity 1
A month；
(c) preparation of internal standard storing solution
Pidolidone -2,4,4-d3 standard items 1mg is taken to be placed in 10mL volumetric flasks, dissolved with 50% methanol aqueous solution,
And constant volume obtains standard reserving solution C, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 year；
It takes L-Aspartic acid -2,3,3-d3 standard items 1mg to be placed in 10mL volumetric flasks, is carried out with 50% methanol aqueous solution molten
Solution, and constant volume obtains standard reserving solution D, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 year；
(d) preparation of internal standard working solution is mixed
Internal standard storing solution C and internal standard storing solution D in appropriate step (c) is taken, is carried out with the methanol aqueous solution of 40-60% dilute
It releases, obtains Pidolidone -2,4 containing 25 μM of concentration, L-Aspartic acid -2,3 of 4-d3 and 25 μM of concentration, in the mixing of 3-d3
Working solution C is marked, and is preserved under the conditions of -80 DEG C, the term of validity is 1 year；
(2) centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, supernatant is taken to obtain serum or blood
It is spare to before analyzing that slurry, above-mentioned serum or blood plasma are placed in the lower preservation of -20 DEG C of freezings；
(3) sample to be tested is handled
(e) then the mixing internal standard working solution for pipetting 10 μ L steps (d) with liquid-transfering gun is added in 1.5mL centrifuge tube
Serum or blood plasma described in 20 μ L step (2), add the protein precipitation reagent of 100 μ L, under the rotating speed of 1500-2000rpm
After vortex mixed 2-5min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, supernatant is obtained, 100 μ are pipetted
L is sample to be tested；
(4) detection of sample to be tested
Above-mentioned steps (e) sample to be tested is detected using efficient liquid phase tandem mass spectrometer, while being obtained above-mentioned to be measured
The Pidolidone of sample, the chromatogram of L-Aspartic acid and internal standard compound, from above-mentioned Pidolidone, L-Aspartic acid and internal standard chromatography
Pidolidone chromatographic peak area, L-Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area can be obtained in figure, by L- paddy
The ratio y of propylhomoserin chromatographic peak area and internal standard compound chromatographic peak area1Substitute into the calibration curve equation y of above-mentioned steps (one)1=a*x1
In+b, the relative concentration x of Pidolidone and internal standard compound in detected sample is obtained by calculation1, internal standard compound is in mixing internal standard work
It is known to make the concentration in liquid, and the concentration of the Pidolidone for waiting for sample is thus calculated；By L-Aspartic acid chromatographic peak
The ratio y of area and internal standard compound chromatographic peak area2Substitute into the calibration curve equation y of above-mentioned steps (one)2=c*x2In+d, pass through
The relative concentration x of L-Aspartic acid and internal standard compound in detected sample is calculated2, internal standard compound is in mixing internal standard working solution
Concentration is known, and the L-Aspartic acid concentration of the sample to be tested is thus calculated, i.e. simultaneous quantitative detects that this waits for test sample
The Pidolidone concentration and L-Aspartic acid concentration of product.
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：In step
(1) respectively contain L- paddy ammonia using the standard working solution A of nine kinds of various concentrations, the standard working solution A of nine kinds of various concentrations in
Acid concentration is 5.000 μM, 10.00 μM, 20.00 μM, 40.00 μM, 80.00 μM, 160.0 μM, 320 μM, 640 μM and 1280 μM；
It is respectively to contain that the standard working solution B of ten kinds of various concentrations, the standard working solution B of ten kinds of various concentrations are used in step (1)
L-Aspartic acid acid concentration be 0.15625 μM, 0.31250uM, 0.62500 μM, 1.2500 μM, 2.5000 μM, 5.0000 μM,
10.000 μM, 20.000 μM, 40.000 μM and 160.00 μM；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：In step
(b) it in, is diluted with 50% methanol aqueous solution；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：It is described heavy
Shallow lake protein reagent is methanol；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：The height
Pot strainer used in effect liquid phase chromatogram instrument is Agilent 1290Infinity inline filter；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：The height
Chromatographic column used in effect liquid phase chromatogram instrument is the Kinetex F5 of Phenomenex companies；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：The height
The column temperature of effect liquid phase chromatogram instrument setting is 30 DEG C；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：The height
Effect liquid phase chromatogram instrument the use of mobile phase is the flow velocity 0.3mL/min containing the pure water that volume ratio is 0.05% formic acid, sample size is
2μL；
A kind of while method that detects Pidolidone and L-Aspartic acid content in blood of the present invention, wherein：It is described to contain
Water is the water content of volume ratio.
Advantageous effect of the present invention：
The method that Pidolidone and L-Aspartic acid content in blood are detected while of the present invention, high specificity, standard
Exactness and high sensitivity, analysis time are short, clinically to diagnose the shortage or excess of Pidolidone and L-Aspartic acid in blood
Provide reliable experimental basis.The method that Pidolidone and L-Aspartic acid content in blood are detected while of the present invention
Pre-treatment is carried out in non-derivative method, simple economy reduces the interference of part human factor；It is interior with isotopic label
Mark so that the identification of target compound is more accurate, eliminates systematic error, and quantitative result is accurate.
Description of the drawings
Fig. 1 is the chemical structural formula of Pidolidone；
Fig. 2 is the chemical structural formula of L-Aspartic acid；
Fig. 3 is the chromatogram of Pidolidone in embodiment Plays solution；
Fig. 4 is the chromatogram of Pidolidone Isotopic Internal Standard in embodiment Plays solution；
Fig. 5 is the chromatogram of L-Aspartic acid in embodiment Plays solution；
Fig. 6 is the chromatogram of L-Aspartic acid Isotopic Internal Standard in embodiment Plays solution；
Fig. 7 is the chromatogram of Pidolidone in serum sample in embodiment；
Fig. 8 is the chromatogram of Pidolidone Isotopic Internal Standard in serum sample in embodiment；
Fig. 9 is the chromatogram of L-Aspartic acid in serum sample in embodiment；
Figure 10 is the chromatogram of L-Aspartic acid Isotopic Internal Standard in serum sample in embodiment；
Below in conjunction with specific embodiments and the drawings, the invention will be further described.
Specific implementation mode
The method that Pidolidone and L-Aspartic acid content in blood are detected while of the invention, it is characterised in that：It is wrapped
Include following steps：
(1) calibration of standard solution A and standard solution B
Pipette the nine kinds of various concentration standard working solution A and 10 μ L mixing internal standard working solutions of 20 μ L respectively with pipettor first
It is respectively placed in the standard solution A for being mixed and made into nine kinds of various concentrations in centrifuge tube, is in rotating speed respectively by above-mentioned standard solution
Under 1500rpm after vortex mixing 1min, 100 μ L of protein precipitation reagent methanol are added, and the mixing that is vortexed in the case where rotating speed is 2000rpm
After 3min, then the high speed centrifugation 5min under the rotating speed of 12000rpm, supernatant is obtained, above-mentioned supernatant is pipetted respectively, utilizes height
Effect liquid phase tandem mass spectrometer is detected above-mentioned supernatant, obtains the standard of the Pidolidone and internal standard compound of nine kinds of various concentrations
The chromatogram of solution A respectively obtains L- from the chromatogram of the standard solution A of the Pidolidone and internal standard compound of each above-mentioned concentration
Glutamic acid chromatographic peak area and internal standard compound chromatographic peak area, respectively with the Pidolidone chromatographic peak area of above-mentioned nine various concentrations
Ordinate y with the ratio of internal standard compound chromatographic peak area as calibration curve equation1, with the L- paddy in above-mentioned standard working solution A
Propylhomoserin concentration and the abscissa x that the ratio of the internal standard compound concentration mixed in internal standard working solution is as calibration curve equation1, will be with
The data of at least three kinds various concentrations obtained by upper detection carry out linear regression, and it is y that fitting, which obtains calibration curve equation,1=a*x1+
B, and obtain weight coefficient a, b, such as：It is y that fitting, which obtains Pidolidone calibration curve equation,1=2.1398*x1+
0.012519；
It pipettes 20 μ L, ten various concentration standard working solution B respectively with pipettor and 10 μ L mixing internal standard working solutions is respectively placed in
The standard solution B of ten kinds of various concentrations is mixed and made into centrifuge tube, by above-mentioned standard solution B respectively in the case where rotating speed is 1500rpm
After vortex mixing 1min, 100 μ L of protein precipitation reagent methanol are added, and in the case where rotating speed is 2000rpm after vortex mixing 3min, then
The high speed centrifugation 5min under the rotating speed of 12000rpm, obtains supernatant, pipettes above-mentioned supernatant respectively, is connected using efficient liquid phase
Mass spectrograph is detected above-mentioned supernatant, obtains the standard working solution B of the L-Aspartic acid and internal standard compound of ten kinds of various concentrations
Chromatogram, respectively obtain L- from the chromatogram of the standard working solution B of the L-Aspartic acid and internal standard compound of each above-mentioned concentration
Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area, with the chromatographic peak face of the L-Aspartic acid of above-mentioned ten kinds of various concentrations
Ordinate y of the ratio of product and internal standard compound chromatographic peak area as calibration curve equation2；With the L- in above-mentioned standard working solution B
Abscissa x of the aspartic acid concentration with the ratio of the internal standard compound concentration mixed in internal standard working solution as calibration curve equation2, will
The data of nine kinds of various concentrations of the above detection gained carry out linear regression, and it is y that fitting, which obtains calibration curve equation,2=c*x2+ d,
And obtain weight coefficient c, d, such as：It is y that fitting, which obtains L-Aspartic acid calibration curve equation,2=1.216*x2+0.008833
Criteria discussed above working solution A is the solution containing Pidolidone, and standard working solution B is to contain L-Aspartic acid
Solution, mixing internal standard working solution is molten containing Pidolidone and L-Aspartic acid mixing Isotopic Internal Standard standard items NSK-A
Liquid；
(a) preparation of standard reserving solution A and standard reserving solution B：
Accurately weighing Pidolidone standard items 6.77mg is placed in 10mL volumetric flasks, is dissolved with 50% methanol aqueous solution,
And constant volume obtains standard reserving solution A, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 month；
Accurately weighing L-Aspartic acid standard items 4.96mg is placed in 10mL volumetric flasks, is carried out with 50% methanol aqueous solution molten
Solution, and constant volume obtains standard reserving solution B, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 month；
(b) preparation of standard working solution A and standard working solution B
Appropriate step (a) Plays storing solution A is taken, is diluted, is obtained containing L- paddy with the methanol aqueous solution of 40-60%
A concentration of 5.000 μM, 10.00 μM, 20.00 μM, 40.00 μM, 80.00 μM, 160.0 μM, 320 μM, 640 μM and 1280 μM of propylhomoserin
Nine kinds of standard working solution A, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
Appropriate step (a) Plays storing solution B is taken, is diluted, is obtained containing L- days with the methanol aqueous solution of 40-60%
Winter propylhomoserin is 0.15625 μM a concentration of, 0.31250 μM, 0.62500 μM, 1.2500 μM, 2.5000 μM, 5.0000 μM, 10.000 μ
M, 20.000 μM, 40.000 μM and 160.00 μM of standard working solution B, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
(c) preparation of internal standard storing solution
Pidolidone -2,4,4-d3 standard items 1mg is taken to be placed in 10mL volumetric flasks, dissolved with 50% methanol aqueous solution,
And constant volume obtains standard reserving solution C, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 year；
It takes L-Aspartic acid -2,3,3-d3 standard items 1mg to be placed in 10mL volumetric flasks, is carried out with 50% methanol aqueous solution molten
Solution, and constant volume obtains standard reserving solution D, and preserved under the conditions of -80 DEG C in 10mL, the term of validity is 1 year；
(d) preparation of internal standard working solution is mixed
Internal standard storing solution C and internal standard storing solution D in appropriate step (c) is taken, is carried out with the methanol aqueous solution of 40-60% dilute
It releases, obtains Pidolidone -2,4 containing 25 μM of concentration, L-Aspartic acid -2,3 of 4-d3 and 25 μM of concentration, in the mixing of 3-d3
Working solution C is marked, and is preserved under the conditions of -80 DEG C, the term of validity is 1 year；
(2) centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, supernatant is taken to obtain serum or blood
It is spare to before analyzing that slurry, above-mentioned serum or blood plasma are placed in the lower preservation of -20 DEG C of freezings；
(3) sample to be tested is handled
(e) then the mixing internal standard working solution for pipetting 10 μ L steps (d) with liquid-transfering gun is added in 1.5mL centrifuge tube
Serum or blood plasma described in 20 μ L step (2), add the protein precipitation reagent of 100 μ L, are vortexed under the rotating speed of 2000rpm
After mixing 3min, then the high speed centrifugation 5min under the rotating speed of 12000rpm, supernatant is obtained, it is sample to be tested to pipette 100 μ L；
(4) detection of sample to be tested
Above-mentioned steps (e) sample to be tested is detected using efficient liquid phase tandem mass spectrometer, while being obtained above-mentioned to be measured
The Pidolidone of sample, the chromatogram of L-Aspartic acid and internal standard compound, from above-mentioned Pidolidone, L-Aspartic acid and internal standard chromatography
Can be obtained in figure Pidolidone chromatographic peak area, L-Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area (such as Fig. 3,
Fig. 4, Fig. 5 and chromatographic peak area shown in fig. 6), by the ratio y of Pidolidone chromatographic peak area and internal standard compound chromatographic peak area1
Substitute into the calibration curve equation y of above-mentioned steps (one)1=a*x1In+b, such as：y1=2.1398*x1+ 0.012519, pass through meter
Calculation obtains the relative concentration x of Pidolidone and internal standard compound in detected sample1, concentration of the internal standard compound in mixing internal standard working solution
It is known, the concentration of the Pidolidone for waiting for sample is thus calculated；By L-Aspartic acid chromatographic peak area and internal standard compound
The ratio y of chromatographic peak area2Substitute into the calibration curve equation y of above-mentioned steps (one)2=c*x2In+d, such as：y2=1.216*x2+
0.008833, the relative concentration x of L-Aspartic acid and internal standard compound in detected sample is obtained by calculation2, internal standard compound mixing
Concentration in internal standard working solution is known, and the L-Aspartic acid concentration of the sample to be tested, i.e. simultaneous quantitative is thus calculated
Detect the Pidolidone concentration and L-Aspartic acid concentration of the sample to be tested.
The Liquid Chromatography-Tandem Mass Spectrometry determination condition is as follows：
Chromatographic column：Kinetex F5,2.6 μm, 4.6mm × 100mm (Phenomenex companies),
Poroshell 120PFP, 2.7 μm, 3.0mm × 100mm (Agilent companies)；
Pot strainer：Agilent 1290Infinity inline filter(0.3μm)；
Column temperature：30℃；
Mobile phase：Pure water (formic acid for being 0.05% containing volume ratio)；
Flow velocity：0.3mL/min；
Analysis time：6min；
Sample size：2μL；
| Substance title | Parent ion | Daughter ion | Retention time (min) | Dwell | Fragmentor | CE | CAV |
| L-Glu | 148 | 84 | 5.03 | 100 | 80 | 18 | 3 |
| L-Asp | 134 | 88 | 5.17 | 100 | 50 | 7 | 4 |
| L-Glu-IS | 151 | 87 | 5.03 | 100 | 80 | 18 | 3 |
| L-Asp-IS | 137 | 91 | 5.17 | 100 | 50 | 7 | 4 |
1 tandem mass spectrum condition of table
Data acquisition modes：MRM (multiple-reaction monitoring pattern)；
Atomization gas temperature：350℃；
Atomization gas air-flow：10L/min
Atomization air pressure：40psi；
Capillary voltage：3.5kV；
Ion source：Electric spray ion source；
Ionization mode：Positive ion mode.
Technical method demonstration is as follows in the present embodiment：
One, the linear relationship and quantitative limit of this method
By the Pidolidone and L-Aspartic acid standard working solution of each concentration of 20 μ l of above-mentioned preparation, it is separately added into 10
μ l mixing internal standard working solutions and 100 μ l methanol, mixing sample introduction are measured from low to high by the present embodiment determination condition concentration,
It is mapped with one concentration of quota ion chromatographic peak area, obtains standard curve, the results showed that the line of Pidolidone and L-Aspartic acid
Property range and quantitative limit are as follows：
(1) detection limit (LOD)：Pidolidone is 0.116 μM；L-Aspartic acid is 0.156 μM.
(2) quantitative limit (LOQ)：Pidolidone is 0.386 μM；L-Aspartic acid is 0.156 μM.
(3) range of linearity：
Pidolidone is linear good within the scope of 5.000 μM to 1280 μM, coefficient R2＞ 0.995；
L-Aspartic acid is linear good within the scope of 0.15625 μM to 160.00 μM, coefficient R2＞ 0.990.
Two, the rate of recovery and precision of this method
It takes Pidolidone and L-Aspartic acid standard working solution to be configured to high, medium and low 3 kinds of concentration respectively and carries out sample-adding recycling
Rate and Precision Experiment are measured by the present embodiment method, and replicate analysis measures 6 batches, Pidolidone and L-Aspartic acid
The rate of recovery and precision respectively such as table 2 and table 3.Pidolidone averagely returning within the scope of 3 basic, normal, high pitch-based spheres
Yield is 100.20%~104.37%, and relative standard deviation is 1.35%~4.10%, the results are shown in Table 2.L-Aspartic acid exists
Average recovery rate within the scope of 3 basic, normal, high pitch-based spheres is 100.18%~104.83%, and relative standard deviation is
0.56%~6.61%, it the results are shown in Table 3.
| Mark-on amount | 13.18μg/mL | 94.38μg/mL | 376.8μg/mL |
| Average recovery rate | 100.20% | 104.37% | 103.84% |
| Precision RSD | 1.35% | 4.10% | 3.87% |
Table 2.L- glutamic acid sample recovery rate and precision
| Mark-on amount | 1.25μg/mL | 10.2μg/mL | 40.5μg/mL |
| Average recovery rate | 100.18% | 101.19% | 104.83% |
| Precision RSD | 0.56% | 6.61% | 4.55% |
Table 3.L- aspartic acids sample recovery rate and precision
In summary verification test, the rate of recovery of the present embodiment, all technicals such as detection limit and precision meet
It is required that method detects Pidolidone and L-Aspartic acid in blood simultaneously, reproducibility is good, and sample recovery rate is good, to
The accuracy of testing result is improved, systematic error is eliminated.
In the present embodiment in standard solution, Pidolidone chromatogram is as shown in Figure 3；The color of Pidolidone Isotopic Internal Standard
Spectrogram is as shown in Figure 4；The chromatogram of L-Aspartic acid is as shown in Figure 5；The chromatogram of L-Aspartic acid Isotopic Internal Standard such as Fig. 6 institutes
Show；In serum sample, Pidolidone chromatogram is as shown in Figure 7；The chromatogram of Pidolidone Isotopic Internal Standard is as shown in Figure 8；L-
The chromatogram of aspartic acid is as shown in Figure 9；The chromatogram of L-Aspartic acid Isotopic Internal Standard is as shown in Figure 10.It can by Fig. 3-10
See, the retention time of Pidolidone and L-Aspartic acid is consistent with its standard working solution, and this method is with isotopic label
Internal standard compound so that the identification of target compound is more accurate, and analysis time is short, interference is small, and interior scalar quantity appropriate specificity is strong, accurate
Exactness and high sensitivity.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
It encloses and is defined, under the premise of not departing from design spirit of the present invention, technical side of the those of ordinary skill in the art to the present invention
The various modifications and improvement that case is made should all be fallen into the protection domain of claims of the present invention determination.
Claims (9)
1. method that is a kind of while detecting Pidolidone and L-Aspartic acid content in blood, it is characterised in that：It includes following
Step：
(1) calibration of standard solution A and standard solution B
Pipette 20 μ L at least three kinds of various concentration standard working solution A and 10 μ L mixing internal standard working solutions point respectively with pipettor first
It is not placed in centrifuge tube and is mixed and made into the standard solution A of at least three kinds various concentrations, be in rotating speed respectively by above-mentioned standard solution
Under 1500-2000rpm after vortex mixing 1-2min, 100 μ L of protein precipitation reagent are added, and in the case where rotating speed is 1500-2000rpm
After vortex mixing 2-4min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, supernatant is obtained, is pipetted respectively
Above-mentioned supernatant is detected above-mentioned supernatant using efficient liquid phase tandem mass spectrometer, obtains at least three kinds of various concentrations
The chromatogram of the standard solution A of Pidolidone and internal standard compound, it is molten from the Pidolidone of each above-mentioned concentration and the standard of internal standard compound
Pidolidone chromatographic peak area and internal standard compound chromatographic peak area are respectively obtained in the chromatogram of liquid A, respectively with above-mentioned at least three
The Pidolidone chromatographic peak area of various concentration and ordinate of the ratio of internal standard compound chromatographic peak area as calibration curve equation
y1, made with the ratio of the internal standard compound concentration mixed in internal standard working solution with the Pidolidone concentration in above-mentioned standard working solution A
For the abscissa x of calibration curve equation1, the data of at least three kinds various concentrations of the above detection gained are subjected to linear regression,
It is y that fitting, which obtains calibration curve equation,1=a*x1+ b, and obtain weight coefficient a, b；
It pipettes 20 μ L at least three kinds of various concentration standard working solution B respectively with pipettor and 10 μ L mixing internal standard working solutions is set respectively
It is mixed and made into the standard solution B of at least three kinds various concentrations in centrifuge tube, is in rotating speed respectively by above-mentioned standard solution B
Under 1500-2000rpm after vortex mixing 1-2min, 100 μ L of protein precipitation reagent are added, and in the case where rotating speed is 1500-2000rpm
After vortex mixing 2-4min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, supernatant is obtained, is pipetted respectively
Above-mentioned supernatant is detected above-mentioned supernatant using efficient liquid phase tandem mass spectrometer, obtains at least three kinds of various concentrations
The chromatogram of the standard working solution B of L-Aspartic acid and internal standard compound, from the L-Aspartic acid of each above-mentioned concentration and internal standard compound
L-Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area are respectively obtained in the chromatogram of standard working solution B, with it is above-mentioned extremely
The chromatographic peak area of the L-Aspartic acid of few three kinds of various concentrations is with the ratio of internal standard compound chromatographic peak area as standard curve side
The ordinate y of journey2；With in above-mentioned standard working solution B L-Aspartic acid concentration and the internal standard compound mixed in internal standard working solution it is dense
Abscissa x of the ratio of degree as calibration curve equation2, the data of at least three kinds various concentrations of the above detection gained are carried out
Linear regression, it is y that fitting, which obtains calibration curve equation,2=c*x2+ d, and obtain weight coefficient c, d；
Criteria discussed above working solution A is the solution containing Pidolidone, and standard working solution B is to contain the molten of L-Aspartic acid
Liquid, mixing internal standard working solution are the mixed solution containing Pidolidone Yu L-Aspartic acid Isotopic Internal Standard；
(a) preparation of standard reserving solution A and standard reserving solution B：
It is accurate to weigh Pidolidone standard items 5mg and be placed in 10mL volumetric flasks, dissolved with 50% methanol aqueous solution, and constant volume in
10mL obtains standard reserving solution A, and is preserved under the conditions of -80 DEG C, and the term of validity is 1 month；
Accurately weighing L-Aspartic acid standard items 5mg is placed in 10mL volumetric flasks, is dissolved with 50% methanol aqueous solution, and constant volume
In 10mL, standard reserving solution B is obtained, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
(b) preparation of standard working solution A and standard working solution B
Appropriate step (a) Plays storing solution A is taken, is diluted, is obtained containing 5 μM -1280 with the methanol aqueous solution of 40-60%
The standard working solution A of the Pidolidone of μM concentration, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
Appropriate step (a) Plays storing solution B is taken, is diluted, is obtained containing 0.15625 μ with the methanol aqueous solution of 40-60%
The standard working solution B of the L-Aspartic acid of M-160.00M μ concentration, and preserved under the conditions of -80 DEG C, the term of validity is 1 month；
(c) preparation of internal standard storing solution
Pidolidone -2,4,4-d3 standard items 1mg is taken to be placed in 10mL volumetric flasks, dissolved with 50% methanol aqueous solution, and fixed
It is dissolved in 10mL, obtains standard reserving solution C, and preserved under the conditions of -80 DEG C, the term of validity is 1 year；
L-Aspartic acid -2,3,3-d3 standard items 1mg is taken to be placed in 10mL volumetric flasks, dissolved with 50% methanol aqueous solution, and
Constant volume obtains standard reserving solution D, and preserved under the conditions of -80 DEG C in 10mL, and the term of validity is 1 year；
(d) preparation of internal standard working solution is mixed
Internal standard storing solution C and internal standard storing solution D in appropriate step (c) is taken, is diluted, is obtained with the methanol aqueous solution of 40-60%
To Pidolidone -2,4 containing 25 μM of concentration, L-Aspartic acid -2,3 of 4-d3 and 25 μM of concentration, the mixing internal standard work of 3-d3
Make liquid C, and preserved under the conditions of -80 DEG C, the term of validity is 1 year；
(2) centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, supernatant is taken to obtain serum or blood plasma,
It is spare to before analyzing that above-mentioned serum or blood plasma are placed in the lower preservation of -20 DEG C of freezings；
(3) sample to be tested is handled
(e) then 20 μ L are added in 1.5mL centrifuge tube in the mixing internal standard working solution for pipetting 10 μ L steps (d) with liquid-transfering gun
Serum described in step (2) or blood plasma add the protein precipitation reagent of 100 μ L, are vortexed under the rotating speed of 1500-2000rpm
After mixing 2-5min, then the high speed centrifugation 4-6min under the rotating speed of 10000-15000rpm, supernatant is obtained, pipettes 100 μ L i.e.
For sample to be tested；
(4) detection of sample to be tested
Above-mentioned steps (e) sample to be tested is detected using efficient liquid phase tandem mass spectrometer, while obtaining above-mentioned sample to be tested
Pidolidone, L-Aspartic acid and internal standard compound chromatogram, from above-mentioned Pidolidone, L-Aspartic acid and internal standard chromatogram
Pidolidone chromatographic peak area, L-Aspartic acid chromatographic peak area and internal standard compound chromatographic peak area can be obtained, by Pidolidone
The ratio y of chromatographic peak area and internal standard compound chromatographic peak area1Substitute into the calibration curve equation y of above-mentioned steps (one)1=a*x1+b
In, the relative concentration x of Pidolidone and internal standard compound in detected sample is obtained by calculation1, internal standard compound is in mixing internal standard work
Concentration in liquid is known, and the concentration of the Pidolidone for waiting for sample is thus calculated；By L-Aspartic acid chromatographic peak face
The ratio y of product and internal standard compound chromatographic peak area2Substitute into the calibration curve equation y of above-mentioned steps (one)2=c*x2In+d, pass through meter
Calculation obtains the relative concentration x of L-Aspartic acid and internal standard compound in detected sample2, internal standard compound is dense in mixing internal standard working solution
Degree is known, and the L-Aspartic acid concentration of the sample to be tested is thus calculated, i.e. simultaneous quantitative detects the sample to be tested
Pidolidone concentration and L-Aspartic acid concentration.
2. the method as described in claim 1 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：It is respectively using the standard working solution A, the standard working solution A of nine kinds of various concentrations of nine kinds of various concentrations in step (1)
A concentration of 5.000 μM, 10.00 μM, 20.00 μM, 40.00 μM, 80.00 μM, 160.0 μM, 320 μM, 640 μM containing Pidolidone
With 1280 μM；The standard working solution B, the standard working solution B of ten kinds of various concentrations of ten kinds of various concentrations are used in step (1)
Respectively contain L-Aspartic acid acid concentration be 0.15625 μM, 0.31250 μM, 0.62500 μM, 1.2500 μM, 2.5000 μM,
5.0000 μM, 10.000 μM, 20.000 μM, 40.000 μM and 160.00 μM.
3. the method as claimed in claim 2 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：In step (b), it is diluted with 50% methanol aqueous solution.
4. the method as claimed in claim 3 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：The protein precipitation reagent is methanol.
5. the method as claimed in claim 4 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：Pot strainer used in the high performance liquid chromatograph is 1290 Infinity inline filter of Agilent.
6. the method as claimed in claim 4 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：Chromatographic column used in the high performance liquid chromatograph is the Kinetex F5 of Phenomenex companies.
7. the method as claimed in claim 4 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：The column temperature of the high performance liquid chromatograph setting is 30 DEG C.
8. the method as claimed in claim 4 for detecting Pidolidone and L-Aspartic acid content in blood simultaneously, feature exist
In：The high performance liquid chromatograph using mobile phase be containing the pure water that volume ratio is 0.05% formic acid, flow velocity 0.3mL/
Min, sample size are 2 μ L.
9. being detected while as described in claim 4-8 any claims in blood Pidolidone and L-Aspartic acid content
Method, it is characterised in that：The water content is the water content of volume ratio.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114577943A (en) * | 2022-03-08 | 2022-06-03 | 山东省食品药品检验研究院 | Method for indirectly measuring infant formula milk powder and modulating aspartic acid and glutamic acid in milk powder |
| CN114981653A (en) * | 2020-01-17 | 2022-08-30 | 沃特世科技公司 | Low binding surface for peptide mapping analysis |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101354385A (en) * | 2008-04-25 | 2009-01-28 | 广东中烟工业有限责任公司 | Method for simultaneously analyzing twenty amino acids in tobacco and product thereof |
| CN101430307A (en) * | 2008-12-18 | 2009-05-13 | 浙江大学 | Method for simultaneously analyzing amino acid and organic acid metabolite spectrum |
| US20130295558A1 (en) * | 2012-05-07 | 2013-11-07 | National Chung Cheng University | Method of detecting ethylated thymidine dna adducts |
| CN105866315A (en) * | 2016-06-14 | 2016-08-17 | 广西中烟工业有限责任公司 | Measuring method for amino acid in electronic cigarette smoke liquid |
| CN106442758A (en) * | 2016-08-31 | 2017-02-22 | 陈大为 | Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode |
-
2018
- 2018-05-17 CN CN201810476876.1A patent/CN108426961A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101354385A (en) * | 2008-04-25 | 2009-01-28 | 广东中烟工业有限责任公司 | Method for simultaneously analyzing twenty amino acids in tobacco and product thereof |
| CN101430307A (en) * | 2008-12-18 | 2009-05-13 | 浙江大学 | Method for simultaneously analyzing amino acid and organic acid metabolite spectrum |
| US20130295558A1 (en) * | 2012-05-07 | 2013-11-07 | National Chung Cheng University | Method of detecting ethylated thymidine dna adducts |
| CN105866315A (en) * | 2016-06-14 | 2016-08-17 | 广西中烟工业有限责任公司 | Measuring method for amino acid in electronic cigarette smoke liquid |
| CN106442758A (en) * | 2016-08-31 | 2017-02-22 | 陈大为 | Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode |
Non-Patent Citations (6)
| Title |
|---|
| EDWARD TAKACH 等: "High-throughput quantitation of amino acids in rat and mouse biological analysis", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| JUN QU 等: "Validated Quantitation of Underivatized Amino Acids in Human Blood Samples by Volatile Ion-Pair Reversed-Phase Liquid Chromatography Coupled to Isotope Dilution Tandem Mass Spectrometry", 《ANAL. CHEM.》 * |
| W.A. HUUB WATERVAL 等: "Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism", 《CLINICA CHIMICA ACTA》 * |
| 张轶雯 等: "串联质谱法测定血清中20种游离氨基酸含量", 《检验医学与临床》 * |
| 郑重 等: "高效液相色谱-串联质谱法直接定量分析植物酵素中多种氨基酸成分", 《色谱》 * |
| 黄翼飞 等: "液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸", 《色谱》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114981653A (en) * | 2020-01-17 | 2022-08-30 | 沃特世科技公司 | Low binding surface for peptide mapping analysis |
| 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 |
| CN114577943A (en) * | 2022-03-08 | 2022-06-03 | 山东省食品药品检验研究院 | Method for indirectly measuring infant formula milk powder and modulating aspartic acid and glutamic acid in milk powder |
| CN114577943B (en) * | 2022-03-08 | 2024-01-23 | 山东省食品药品检验研究院 | Method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder |
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