CN101581703A - Method for single-time measuring ten amino bioactivators in organism - Google Patents
Method for single-time measuring ten amino bioactivators in organism Download PDFInfo
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- CN101581703A CN101581703A CNA2008100433695A CN200810043369A CN101581703A CN 101581703 A CN101581703 A CN 101581703A CN A2008100433695 A CNA2008100433695 A CN A2008100433695A CN 200810043369 A CN200810043369 A CN 200810043369A CN 101581703 A CN101581703 A CN 101581703A
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Abstract
The invention belongs to the field of biochemistry detection, in particular to the measurement of bioactive molecules of neuroendocrine immunological network, particularly to a measurement method of amino bioactivators in organism (such as blood or brain tissue), in particular discloses a method for the single-time measuring of ten amino bioactivators in organism, which comprises: taking dansyl chloride as derivative reagent to perform single-time measuring on ten amino bioactivators such as epinephrine (E), noradrenalin (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), histamine (HA), gluetamic acid (Glu), aspartic acid (Asp), Gamma-aminobutyric acid (GABA), glycin (Gly) and trilute (T3) within 75min. The method is simple and rapid, can sensitively measure the concentrations of the ten amino bioactivators in organism fluid, and is suitable to the research requiring to completely analyze the ten amino bioactivators in the organism.
Description
Technical field
The invention belongs to field of biochemistry detection, particularly relate to the mensuration of neuroendocrine-immune network bioactive molecule, especially the assay method of (as blood or brain tissue) amino class bioactivator in the biosome.
Background technology
The integrally-regulated effect and the neuroendocrine-immune network of traditional Chinese medicine are closely related, and adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T
3) to wait ten kinds of amino class bioactivators be for the research neuroendocrine-immune network significant factor.
Adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T
3) wait in the molecular formula of ten kinds of amino class bioactivators amino is all arranged, dansyl chloride can form the derivant with fluorescence with the amino compound reaction, can be used for the mensuration of trace activity substance.Be that derivative reagent is measured in the blood some amino or certain several amino acid whose document all has report both at home and abroad with the dansyl chloride, but with amino acids in the blood and monoamine transmitters together, be that the chromatographic condition that derivative reagent detects does not appear in the newspapers both at home and abroad with the dansyl chloride; With amino acids and monoamine transmitters, histamine, trilute together, be that the chromatographic condition that derivative reagent detects does not appear in the newspapers both at home and abroad with the dansyl chloride.
With the adrenaline in the neuroendocrine-immune network (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T
3) wait ten kinds of amino class bioactivators according to the conventional sense method measure: detect adrenaline (E), norepinephrine (NE), dopamine catecholamines neurotransmitters such as (DA) as high performance liquid chromatography-electrochemical; With o-phthalaldehyde(OPA) is that fluorescent derivatizing agent high performance liquid chromatography fluorescence detection is measured amino acid neurotransmitter; Measure trilute etc. with putting the method for exempting from,, therefore, need the detected sample size can be bigger, the increase experimental cost on the one hand because method is different; On the other hand, since different detection methods, sample pre-treatments step difference, the principle difference of detection, in the time of can causing unified Analysis to detect the variation of index, the systematic error of experiment increases.
Summary of the invention
Technical matters to be solved by this invention provides the method for ten kinds of amino class bioactivators in a kind of single-time measurement biosome.It is derivative reagent single-time measurement adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T that the present invention adopts dansyl chloride
3) wait ten kinds of amino class bioactivators, make the mensuration of amino class bioactivators such as adrenaline in the biosome convenient fast.
In order to solve the problems of the technologies described above, the method for ten kinds of amino class bioactivators comprises the steps: in the single-time measurement biosome provided by the invention
Biological fluid after deproteinized is handled, is added KHCO
3After solution is transferred pH value, add again with transfer PH after liquor capacity identical, concentration is that the dansyl chloride of 2-10mg/ml carries out derivative reaction, temperature of reaction is 80 ℃ of water-baths, the reaction time is 20 minutes;
Utilize high performance liquid chromatograph and fluorescence detector to finish to adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T
3) detection of ten kinds of amino class bioactivators, wherein,
Chromatographic condition is:
Chromatographic column: Hypersil ODS (C18) chromatographic column, model 250 * 4.6mm, 5 μ m;
Moving phase: Mobile phase B is a methyl alcohol: acetonitrile=2: 3; Mobile phase A is the acetate buffer of 20mmol/l, wherein adds the perfluorooctane sulfonate of 3mmol/l, the Na-EDTA of 0.1mmol/l, and transfer to pH value 4.8 with NaOH, use the 0.45um filtering with microporous membrane;
Flow velocity: 1mlmin
-1
Column temperature: 30 ℃;
The excitation wavelength of fluorescence detector is 350nm, and emission wavelength is 510nm.
Among the present invention, adopt gradient elution in the liquid chromatography, the condition such as the following table of gradient elution are listed.
| Time | Mobile phase B (%) | Mobile phase A (%) |
| 0～ |
10～20→35～45 | 90～80→65～55 |
| 10～30min | 35～45 | 65～55 |
| 30～40min | 35-45→75～85 | 65～55→25～15 |
| 40～50min | 75～85 | 25～15 |
| 50～70min | 75～85→85～95 | 25～15→10～5 |
| 70～80min | 85～95 | 15～5 |
With respect to prior art, the inventive method is with adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly) and trilute (T
3) these ten kinds of materials detected in 75 minutes in same chromatographic system, greatly reduced the cost of experiment, had saved the man power and material; The inventive method is particularly evident for the less situation effect of biological specimen amounts such as zoopery; The inventive method has also reduced the systematic error that different detection methods cause.The inventive method can be used for the analysis and research of ten kinds of amino class bioactivators such as adrenaline in the neuroendocrine-immune network, help to illustrate in the neuroendocrine-immune network between ten kinds of amino class bioactivators such as adrenaline and with the relation of the integrally-regulated effect of Chinese medicine.
Description of drawings
Fig. 1 is a hybrid standard product reaction back collection of illustrative plates in the 0-90min.
Fig. 2 is a blood plasma reaction back collection of illustrative plates (amplification of 45-90min collection of illustrative plates) in the 0-90min.
Embodiment
Below in conjunction with the drawings and specific embodiments, further set forth the present invention.These embodiment are interpreted as only being used to the present invention is described and are not used in restriction protection scope of the present invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modify and fall into claim of the present invention institute restricted portion equally.
The method of ten kinds of amino class bioactivators in the single-time measurement biosome that a preferred embodiment of the present invention proposes, comprise the foundation of the HPLC analytical method of the amino class bioactivator of blood, and measure take certain simply behind the Chinese medicine to the influence of these ten factors in the neuroendocrine-immune network.
1 reagent and equipment.
1.1 instrument
LC-20AB high performance liquid chromatograph (day island proper Tianjin company); RF-10Axl fluorescence detector (day island proper Tianjin company); LC chromatographic work station (Beijing is safe upright); GL-20B high speed freezing centrifuge (Anting Scientific Instrument Factory, Shanghai)
1.2 reagent
Adrenaline (E), norepinephrine (NE), dopamine (DA), five-hydroxy color amine (5-HT), histamine (HA), glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA), glycocoll (Gly), trilute (T
3), thyroid hormone, 4 dihydroxy benzylamine standard items be the Sigma product; Other reagent such as dansyl chloride, methyl alcohol, acetonitrile are chromatographically pure; Water is ultrapure water.
The preparation of 2 samples
2.1 get blood: animal used as test is adopted kunming mice, the cleaning level.Get blank group mouse, anesthesia is the abdominal aorta blood sampling down.Blood is put in the test tube that contains anti-coagulants, and after the mixing, with 3000rpm/min, centrifugal 10min makes with blood cell and separates, and gets faint yellow supernatant and promptly gets blood plasma.After adding 30% trichloroacetic acid in the blood plasma, 10000rpm, 2min gets supernatant after centrifugal 2 times, places the Effendorf pipe ,-20 ℃ of freezing preservations.
2.2 the derivative reaction of blood plasma: get supernatant 100ul in centrifuge tube, add 100ul (2mol/L) KHCO
3, the concentration of 200ul is the dansyl chloride acetone soln of 5mg/ml, mixing places 30min under 80 ℃ of water-baths, takes out the back sample introduction.
3 chromatographic conditions
Chromatographic column: Thermo Hypersil-Keystone C18 ODS (250 * 4.6,5 μ m)
Moving phase: Mobile phase B is a methyl alcohol: acetonitrile=2: 3; Mobile phase A is acetate buffer (20mmol/l), wherein adds perfluorooctane sulfonate (3mmol/l), and Na-EDTA (0.1mmol/l) transfers to pH value 4.8 with NaOH, uses the 0.45um filtering with microporous membrane.
Flow velocity: 1ml/min;
Fluorescence detector: excitation wavelength is 350nm; Emission wavelength is 510nm
Column temperature: 30 ℃;
Sample size: 50ul.
Adopt gradient elution in the liquid chromatography, the condition such as the table 1 of gradient elution are listed.
Table 1. condition of gradient elution
| Time | Mobile phase A (%) | Mobile phase B (%) |
| 0～10min | 15→40 | 85→60 |
| 10～ |
40 | 60 |
| 30～ |
40→80 | 60→20 |
| 40～ |
80 | 20 |
| 50～ |
80→90 | 20→10 |
| 70～80min | 90 | 10 |
The reacted collection of illustrative plates of hybrid standard product as shown in Figure 1 in the 0-90min.
The reacted collection of illustrative plates of blood plasma as shown in Figure 2 in the 0-90min.
4 method performance index
4.1 the typical curve range of linearity and method detection limit
Make the standard series of 0-10.0ug/ml with standard reserving solution, behind fluorescence derivation, sample introduction 50ul, chromatographic resolution, fluorescence detector detects.With the 3 times of pairing generation survey of baseline noise substrate concentrations is the detectability of method.After area under curve and content are taken the logarithm, carry out correlation analysis, be linear dependence.Typical curve and regression equation and detectability see Table 2.Wherein Y represents the logarithm value of area under curve, and X represents the logarithm value of content (ng).
Table 2. method standard regression equation related coefficient and detection limit
| Measure material | The standard regression equation | Related coefficient (r) | Detection limit (ng/ml) |
| Glutamic acid | Y＝3.74+0.92x | 0.992 | 0.79 |
| Aspartic acid | Y＝3.90+0.90x | 0.973 | 0.72 |
| Glycocoll | Y＝4.55+0.83x | 0.994 | 0.22 |
| Aminobutyric acid | Y＝4.77+0.78x | 0.970 | 0.07 |
| Histamine | Y＝2.62+1.19x | 0.979 | 0.92 |
| 5-HT | Y＝3.94+0.65x | 0.947 | 0.96 |
| T3 | Y＝5.21+0.21x | 0.955 | 0.003 |
| NE | Y＝3.37+0.95x | 0.991 | 0.80 |
| E | Y＝4.09+0.73x | 0.983 | 0.68 |
| DA | Y＝4.00+0.76x | 0.979 | 0.35 |
4.2 method accuracy and precision.
4.2.1 accuracy
Under the experiment condition that the present embodiment method is determined, calculating goes out theoretical concentration according to calculated by peak area, and with the comparing of actual concentrations, obtain accuracy, the results are shown in Table 3, by table 3 as seen, the recovery scope of present embodiment method is 86.8%-117.8%, and its precision satisfies the requirement that in-vivo content is analyzed.
4.2.2 precision
Under the experiment condition that the present embodiment method is determined, mixed mark in a few days and is in the daytime respectively measured 5 times, and according to calculated by peak area in a few days and relative standard deviation in the daytime, measurement result sees Table 3, by table 3 as seen, the relative standard deviation scope of present embodiment method is 0.2%-14.4%.Its precision satisfies the requirement that in-vivo content is analyzed.
Accuracy of table 3. method and precision
4.3 for example:
According to the area under curve of each material among Fig. 2,, see Table 4 according to each index determining result in the above-mentioned regression equation calculation mice plasma.
Table 4. mice plasma measurement result
| Measure material | Concentration ng/ml |
| Glutamic acid | 159.2 |
| Aspartic acid | 263.0 |
| Glycocoll | 82.7 |
| Aminobutyric acid | 745.9 |
| Histamine | 532.5 |
| 5-HT | 65.6 |
| T3 | 148.8 |
| NE | 157.7 |
| E | 21.2 |
| DA | 28.1 |
Claims (2)
1, the method for ten kinds of amino class bioactivators in the single-time measurement biosome is characterized in that this method comprises the steps:
Biological fluid after deproteinized is handled, is added KHCO
3After solution is transferred pH value, add again with transfer PH after liquor capacity identical, concentration is that the dansyl chloride of 2-10mg/ml carries out derivative reaction, temperature of reaction is 80 ℃ of water-baths, the reaction time is 20 minutes;
Utilize high performance liquid chromatograph and fluorescence detector to finish the detection of adrenaline in the biological fluid, norepinephrine, dopamine, five-hydroxy color amine, histamine, glutamic acid, aspartic acid, γ-An Jidingsuan, glycocoll and ten kinds of amino class bioactivators of trilute, wherein
Chromatographic condition is:
Chromatographic column: Hypersil ODS (C18) chromatographic column, model 250 * 4.6mm, 5 μ m;
Moving phase: Mobile phase B is a methyl alcohol: acetonitrile=2: 3; Mobile phase A is the acetate buffer of 20mmol/l, wherein adds the perfluorooctane sulfonate of 3mmol/l, the Na-EDTA of 0.1mmol/l, and transfer to pH value 4.8 with NaOH, use the 0.45um filtering with microporous membrane;
Flow velocity: 1mlmin
-1
Column temperature: 30 ℃;
The excitation wavelength of fluorescence detector is 350nm, and emission wavelength is 510nm.
2, method according to claim 1 is characterized in that, adopts gradient elution in the liquid chromatography, and the condition such as the following table of gradient elution are listed.
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| CN102830189A (en) * | 2012-09-19 | 2012-12-19 | 江南大学 | High performance liquid chromatography (HPLC) measurement method of activity of L-Aspartic acid-decarboxylase |
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| CN104062388A (en) * | 2014-05-09 | 2014-09-24 | 广州艾迪康医学检验所有限公司 | Reagent for HPLC detection of concentration of catecholamines in sample |
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| CN107894478A (en) * | 2017-12-18 | 2018-04-10 | 光明乳业股份有限公司 | A kind of method of gamma aminobutyric acid in semi-solid/solid dairy products using liquid chromatographic detection |
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