CN113820437A - Method for removing catecholamine and intermediate metabolites thereof from blood plasma and application - Google Patents
Method for removing catecholamine and intermediate metabolites thereof from blood plasma and application Download PDFInfo
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Abstract
The invention discloses a method for removing catecholamine and intermediate metabolites thereof from blood plasma and application thereof. Specifically, the invention discloses a method for removing epinephrine (E), Norepinephrine (NE), Methoxyepinephrine (MN), Dopamine (DA), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) in plasma. The invention comprises the following steps: the method comprises the steps of firstly treating plasma through a solid phase extraction device, an adsorbent and an ultrafiltration tube, finally detecting through a liquid chromatography tandem mass spectrometry, and comparing the influence of different treatment modes on catecholamine and intermediate metabolites thereof in the plasma, so as to screen out a better treatment mode and obtain a blank plasma matrix. The preparation method of the blank plasma matrix obtained by the invention can be used for preparation, production and application of blank samples in a system, a platform, a detection device or a kit for detecting catecholamine and intermediate metabolites thereof in plasma.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of a blank plasma matrix, and especially relates to a method for removing catecholamine and intermediate metabolites thereof from plasma and application thereof.
Background
Catecholamines include dopamine, norepinephrine and epinephrine, the major metabolites of which are 3-methoxytyramine, methoxynorepinephrine and methoxyepinephrine. The detection of catecholamines and their intermediate metabolites is the cornerstone of the diagnosis of neuroendocrine tumors derived from the neural crest, such as pheochromocytoma, paraganglioma and neuroblastoma.
When catecholamine and intermediate metabolites thereof are clinically detected, a detection sample is plasma, and if a matrix configured with a calibration curve is the same as or similar to a sample to be detected, the accuracy and the stability of an experimental method can be better ensured. Although plasma can be used directly as a matrix for a calibration curve for clinical monitoring, since catecholamines are endogenous metabolites and catecholamines and intermediate metabolites thereof are present in a certain amount in a plasma sample, configuring the calibration curve with untreated plasma results in low detection results of the sample. Therefore, blank plasma with catecholamine and intermediate metabolites removed from plasma is needed, and a calibration curve is configured by using the blank plasma to ensure that the content of the catecholamine and the intermediate metabolites in the detected sample is closer to the true value.
The patent CN112730699A discloses a method and a kit for detecting catecholamine and its metabolites in plasma, the main contents of the patent are the pretreatment method and the detection method of catecholamine and its metabolites, and the preparation reagent of the standard solution is 0.1mol/L hydrochloric acid, which is not plasma, so the deviation between the measured sample result and the true value is large.
The prior patent CN202010012635.9 discloses the preparation and application of a quality control substance of catecholamine substances and metabolites of plasma lyophilized powder, and the patent mainly describes the configuration mode of the catecholamine quality control substance of plasma; the blank plasma contained therein was treated with activated carbon only and was not optimized.
The prior patent CN201710657234.7 discloses a method for preparing blank biological samples, but it results in biological blanks without vitamins, and blanks without catecholamines and their intermediate metabolites.
The prior patent CN202010927571.5 discloses a method for preparing blank plasma matrix and its application, which is mainly suitable for vitamins and antibiotics, and is not suitable for preparing blank samples of catecholamine and its intermediate metabolites.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing a blank plasma matrix and applications thereof. The invention discloses a method for removing epinephrine (E), Norepinephrine (NE), methoxy epinephrine (MN), Dopamine (DA), methoxy norepinephrine (NMN) and 3-methoxytyramine (3-MT) in plasma. The invention comprises the following steps: the method comprises the steps of firstly treating plasma by a solid phase extraction device, an adsorbent, an ultrafiltration tube and the like, finally detecting by a liquid chromatography tandem mass spectrometry method, and comparing the influence of different treatment modes on catecholamine and intermediate metabolites thereof in the plasma, so as to screen out a better treatment mode and obtain a blank plasma matrix. The preparation method of the blank plasma matrix obtained by the invention can be used for preparation, production and application of blank samples in a system, a platform, a detection device or a kit for detecting catecholamine and intermediate metabolites thereof in plasma.
The purpose of the invention is realized by the following technical scheme:
a blank plasma matrix is prepared by subjecting a plasma sample to solid phase extraction, adsorption and ultrafiltration.
As an embodiment of the invention, the plasma sample is firstly subjected to solid phase extraction, then is subjected to adsorption treatment, is centrifuged, is collected to obtain supernatant, is subjected to ultrafiltration treatment, is discarded, and is subjected to fixed solution to obtain blank plasma matrix.
Further, the preparation method of the blank plasma matrix comprises the following steps:
s1, activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
s2, taking 5-6mL of plasma sample, loading the plasma sample into the PEP column, and collecting effluent to obtain blank plasma matrix crude extract subjected to solid phase extraction;
s3, adding 4-6mL of ultrapure water, 0.1-0.2g of activated carbon and 0.01-0.02g of glucan into a centrifuge tube, oscillating, centrifuging at 15000-;
s4, adding the blank plasma matrix crude extract obtained in the step S2 into the drained glucan-coated activated carbon prepared in the step S3, oscillating, centrifuging at 15000-20000rpm for 5-10min, taking supernatant, adding the supernatant into the drained glucan-coated activated carbon, oscillating, further centrifuging at 15000-20000rpm for 5-10min, and collecting supernatant to obtain blank plasma matrix primary liquid;
s5, further placing the blank plasma matrix primary liquid prepared in the step S4 in a 3KD ultrafiltration tube, centrifuging for 15-20min at 10000-14000rpm, and discarding the filtrate; adding 300 plus 400 mu L of ultrapure water into the supernatant in an ultrafiltration tube, uniformly mixing, and centrifuging again at 10000 plus 14000rpm for 15-20 min; the filtrate was discarded and the above steps were repeated several times, after which the initial volume was made up with ultrapure water.
In another embodiment of the present invention, a blood sample is subjected to adsorption treatment, followed by solid phase extraction treatment, centrifugation, and then supernatant is collected, and the supernatant is subjected to ultrafiltration treatment, and the filtrate is discarded, and a blank plasma matrix is obtained after solubilization.
Preferably, the preparation method of the blank plasma matrix comprises the following steps:
s1, adding 4-5mL of ultrapure water, 0.1-0.2g of activated carbon and 0.01-0.02g of glucan into a centrifuge tube, oscillating, centrifuging at 15000-;
s2, taking 5-6mL of plasma sample, adding the plasma sample into the dextran-coated activated carbon drained in the step S1, oscillating, and centrifuging at 15000-20000rpm for 5-10 min; taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, oscillating, and centrifuging at 15000-20000rpm for 5-10 min; collecting the supernatant to obtain crude extract of blank plasma matrix subjected to adsorption treatment;
s3, activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water sequentially, and discarding a collecting solution;
s4, taking the blank plasma matrix crude extract prepared in the step S2, loading the blank plasma matrix crude extract into a PEP column activated in the step S3, and collecting effluent to obtain blank plasma primary liquid;
s5, further placing the blank plasma matrix primary liquid prepared in the step S4 in a 3KD ultrafiltration tube, centrifuging for 15-20min at 10000-14000rpm, and discarding the filtrate; adding 300 plus 400 mu L of ultrapure water into the supernatant in an ultrafiltration tube, uniformly mixing, and centrifuging again at 10000 plus 14000rpm for 15-20 min; the filtrate was discarded and the above steps were repeated several times, after which the initial volume was made up with ultrapure water.
The blank plasma matrix prepared by the method also belongs to the protection scope of the invention.
The application of the blank plasma matrix prepared by the method in the invention in target detection of plasma samples also belongs to the protection scope of the invention.
The target comprises one or more of adrenaline (E), Noradrenaline (NE), Methoxyadrenaline (MN), Dopamine (DA), methoxynoradrenaline (NMN) and 3-methoxytyramine (3-MT).
Compared with the prior art, the invention has the following beneficial effects:
1. by comparing the corresponding value difference of catecholamine and intermediate metabolites thereof in the plasma after the treatment of adsorption, solid-phase extraction, adsorption + solid-phase extraction, solid-phase extraction + adsorption, the obtained plasma after the treatment of solid-phase extraction + adsorption has lower content of catecholamine and intermediate metabolites thereof.
2. Further, based on the plasma after solid phase extraction and adsorption treatment, the influence of ultrafiltration tubes with different specifications and different reagents on the removal of catecholamine and intermediate metabolites in the plasma is compared. It was found that the response values of catecholamines and their intermediate metabolites in plasma after 3KD ultrafiltration tube treatment with ultrapure water were low.
3. Finally, comparing the change of response values of catecholamine and intermediate metabolites in the plasma by comparing whether ultraviolet irradiation exists or not, and preferably selecting the plasma after solid phase extraction, adsorption and 3KD ultrafiltration tube treatment as blank plasma.
4. By treating the plasma in a preferred manner, the response of catecholamines and their intermediary metabolites in the blank plasma is less than 10% of the lowest point response of the calibrator. Can be used for the preparation, production and application of blank samples in catecholamine and intermediate metabolite systems, platforms, detection devices or kits.
Drawings
FIG. 1 is a calibration curve for epinephrine (E) in blank plasma prepared in example 7;
FIG. 2 is a calibration curve for Norepinephrine (NE) in blank plasma prepared in example 7;
FIG. 3 is a calibration curve for Methoxyepinephrine (MN) in blank plasma prepared in example 7;
FIG. 4 is a calibration curve of Dopamine (DA) in blank plasma prepared in example 7;
FIG. 5 is a calibration curve for norepinephrine (NMN) in a blank plasma prepared in example 7;
FIG. 6 is a calibration curve of 3-methoxytyramine (3-MT) in blank plasma prepared in example 7.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Example 1
The treatment method comprises the following steps: solid phase extraction + adsorption.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma extracted in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) and (3) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant, namely a blank matrix for later use.
Example 2
The treatment method comprises the following steps: adsorption + solid phase extraction
1) Adding 4.8mL of ultrapure water, 0.12g of active carbon and 0.012g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated active carbon;
2) adding 6mL of plasma into the drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
3) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min); collecting the supernatant;
4) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water in sequence, and discarding a collecting solution; and then 5mL of supernatant after adsorption in the step 3) is taken and loaded into a PEP column, and effluent liquid is collected to be a blank matrix to be detected.
Comparative example 1
The treatment method comprises the following steps: adsorption
The processing steps are as follows:
1) adding 4.8mL of ultrapure water, 0.12g of active carbon and 0.012g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated active carbon;
2) adding 6mL of plasma into the drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
3) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min); collecting the supernatant; obtaining a blank matrix to be tested.
Comparative example 2
The treatment method comprises the following steps: solid phase extraction
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected; the blank matrix is prepared for later use.
Example 3
Effect testing
Blank plasma samples treated in examples 1-2 and comparative examples 1 and 2 were selected and detected by liquid mass spectrometry:
comparing the influence of different treatment modes and treatment sequences of adsorption, solid-phase extraction, adsorption + solid-phase extraction and solid-phase extraction + adsorption on the content of catecholamine and intermediate metabolites in blood plasma:
TABLE 1 catecholamine and intermediate metabolite response values in plasma after different modes of treatment
Through liquid mass spectrum detection, the content difference of 6 compounds is comprehensively analyzed, solid phase extraction is obtained, then activated carbon is used for adsorption, and the content of catecholamine and intermediate metabolites thereof in blank plasma obtained through treatment is low.
Example 4
The treatment method comprises the following steps: solid phase extraction, adsorption and ultrafiltration tubes.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma adsorbed in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant for later use;
6) taking 0.5mL of the plasma adsorbed in the step 5) in an ultrafiltration tube with 10KD, centrifuging for 15min at 14000rpm, and discarding the filtrate; adding 400 μ L of Dithiothreitol (DTT) aqueous solution into the supernatant, mixing well, centrifuging again at 14000rpm for 15 min; discarding the filtrate, and repeating the above steps for 2 times; and finally, fixing the volume to the initial volume by using a DTT solution to obtain a blank plasma sample to be detected.
Example 5
The treatment method comprises the following steps: solid phase extraction, adsorption and ultrafiltration tubes.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma adsorbed in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant for later use;
6) taking 0.5mL of the plasma adsorbed in the step 5) in an ultrafiltration tube with 10KD, centrifuging for 15min at 14000rpm, and discarding the filtrate; adding 400 μ L of ultrapure water into the supernatant, mixing uniformly, and centrifuging again at 14000rpm for 15 min; discarding the filtrate, and repeating the above steps for 2 times; and finally, fixing the volume to the initial volume by using ultrapure water to obtain a blank plasma sample to be detected.
Example 6
The treatment method comprises the following steps: solid phase extraction, adsorption and ultrafiltration tubes.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma adsorbed in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant for later use;
6) taking 0.5mL of the plasma adsorbed in the step 5) in an ultrafiltration tube with 3KD, centrifuging for 15min at 14000rpm, and discarding the filtrate; adding 300 μ L DTT solution into the supernatant, mixing well, centrifuging again at 14000rpm for 15 min; discarding the filtrate, and repeating the above steps for 2 times; and finally, fixing the volume to the initial volume by using a DTT solution to obtain a blank plasma sample to be detected.
Example 7
The treatment method comprises the following steps: solid phase extraction, adsorption and ultrafiltration tubes.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma adsorbed in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant for later use;
6) taking 0.5mL of the plasma adsorbed in the step 5) in an ultrafiltration tube with 3KD, centrifuging for 15min at 14000rpm, and discarding the filtrate; adding 300 μ L of ultrapure water into the supernatant, mixing uniformly, and centrifuging again at 14000rpm for 15 min; discarding the filtrate, and repeating the above steps for 2 times; and finally, fixing the volume to the initial volume by using ultrapure water to obtain a blank plasma sample to be detected.
Example 8
And selecting blank plasma samples treated in examples 4-7, detecting by a liquid mass spectrometry, and screening out a better treatment mode and an ultrafiltration tube.
Table 2 examples 4-7 catecholamines and their intermediary metabolite responses in plasma after different ultrafiltration tube and reagent treatments
Through liquid phase mass spectrum detection, response differences of 6 compounds are comprehensively compared, a 3KD ultrafiltration tube is used, and the obtained blank plasma has a good effect through a treatment mode of ultrapure water constant volume.
Example 9
The treatment method comprises the following steps: solid phase extraction + adsorption + ultrafiltration tube + UV.
The processing steps are as follows:
1) activating a functionalized polystyrene/divinylbenzene extraction column (PEP column) by using methanol and water, and discarding a collecting solution;
2) then 6mL of plasma is taken and loaded into a PEP column, and effluent is collected;
3) adding 4mL of ultrapure water, 0.1g of activated carbon and 0.01g of glucan into a centrifugal tube, oscillating, centrifuging at 18000rpm for 5min, removing supernatant, and leaving glucan-coated activated carbon;
4) adding 5mL of the plasma adsorbed in the step 2) into drained glucan-coated activated carbon, shaking and centrifuging (18000rpm for 5 min);
5) taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, shaking, centrifuging (18000rpm for 5min), and collecting the supernatant for later use;
6) taking 0.5mL of the treated plasma in a 3KD ultrafiltration tube, centrifuging for 15min at 14000rpm, and discarding the filtrate; adding 400 μ L of ultrapure water into the supernatant, mixing uniformly, and centrifuging again at 14000rpm for 15 min; discarding the filtrate, repeating the above steps for 2 times, and finally metering the volume to the initial volume by using ultrapure water;
7) and (3) taking the plasma treated by the ultrafiltration tube, and irradiating for 10min by ultraviolet to obtain a blank plasma sample to be detected.
Example 10
Effect testing
Blank plasma samples after treatment of example 7 and example 9 were taken and tested by liquid mass spectrometry: comparison of the effect of uv irradiation on blank plasma:
TABLE 3 Effect of UV irradiation on catecholamines and their intermediate metabolites in plasma
By comparing the response values of catecholamines and their intermediate metabolites in the plasma after UV treatment with those in the plasma without UV treatment, it was found that the contents of catecholamines and their intermediate metabolites in the plasma could not be reduced after UV treatment. Therefore, the optimal combination for removing catecholamine and intermediate metabolites thereof in blood plasma is selected as follows: solid phase extraction + adsorption +3KD ultrafiltration tube.
Example 11
Comparative example 7 response of blank plasma prepared with the preferred treatment combination (solid phase extraction + adsorption +3KD ultrafiltration tube) to catecholamines and their intermediate metabolites at the lowest point of calibrator concentration:
table 4 comparison of the lowest point of concentration of blank plasma and calibrator
The response of catecholamines and their intermediary metabolites in the blank plasma after optimal condition treatment is less than 10% of the lowest point response of the calibrator concentration.
The blank plasma obtained in the processing mode of the embodiment 7 is prepared into a calibrator of catecholamine and intermediate metabolites thereof in the plasma, and a calibration curve is established by adopting an isotope internal standard quantitative method, taking the concentration ratio of a standard substance as an x axis and taking the peak area ratio of the standard substance and the internal standard substance as a y axis; the calibration curves obtained are shown in fig. 1-6: the linear fitting equation of catecholamine and intermediate metabolites thereof in respective concentration ranges is good in linearity, and correlation coefficients are all above 0.99 at R.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (8)
1. A method for preparing a blank plasma matrix, which is characterized by comprising the following steps: after the plasma sample is processed by solid phase extraction, adsorption and ultrafiltration, a blank plasma matrix is obtained.
2. The method for preparing a blank plasma matrix according to claim 1, wherein: performing solid phase extraction on a plasma sample, then performing adsorption treatment, centrifuging, collecting supernatant, and performing ultrafiltration treatment on the supernatant to obtain a blank plasma matrix.
3. The method for preparing the blank plasma matrix according to claim 2, wherein the method for preparing the blank plasma matrix comprises the following steps:
s1, activating the functionalized polystyrene/divinylbenzene extraction column by using methanol and water, and discarding a collection liquid;
s2, taking 5-6mL of plasma sample, loading the plasma sample into the functionalized polystyrene/divinylbenzene extraction column, and collecting effluent to obtain a blank plasma matrix crude extract subjected to solid phase extraction;
s3, adding 4-6mL of ultrapure water, 0.1-0.2g of activated carbon and 0.01-0.02g of glucan into a centrifuge tube, oscillating, centrifuging at 15000-;
s4, adding the blank plasma matrix crude extract obtained in the step S2 into the drained glucan-coated activated carbon prepared in the step S3, oscillating, centrifuging at 15000-20000rpm for 5-10min, taking supernatant, adding the supernatant into the drained glucan-coated activated carbon, oscillating, further centrifuging at 15000-20000rpm for 5-10min, and collecting supernatant to obtain blank plasma matrix primary liquid;
s5, further placing the blank plasma matrix primary liquid prepared in the step S4 in a 3KD ultrafiltration tube, centrifuging for 15-20min at 10000-14000rpm, and discarding the filtrate; adding 300 plus 400 mu L of ultrapure water into the supernatant in an ultrafiltration tube, uniformly mixing, and centrifuging again at 10000 plus 14000rpm for 15-20 min; the filtrate was discarded and the above steps were repeated several times, after which the initial volume was made up with ultrapure water.
4. The method for preparing a blank blood substrate according to claim 1, wherein: the blood sample is firstly subjected to adsorption treatment and then solid phase extraction treatment, supernatant is collected after centrifugation, and the supernatant is subjected to ultrafiltration treatment to obtain a blank plasma matrix.
5. The method for preparing the blank plasma matrix according to claim 4, wherein the method for preparing the blank plasma matrix comprises the following steps:
s1, adding 4-5mL of ultrapure water, 0.1-0.2g of activated carbon and 0.01-0.02g of glucan into a centrifuge tube, oscillating, centrifuging at 15000-;
s2, taking 5-6mL of plasma sample, adding the plasma sample into the dextran-coated activated carbon drained in the step S1, oscillating, and centrifuging at 15000-20000rpm for 5-10 min; taking the supernatant, adding the supernatant into the drained glucan-coated activated carbon, oscillating, and centrifuging at 15000-20000rpm for 5-10 min; collecting the supernatant to obtain crude extract of blank plasma matrix subjected to adsorption treatment;
s3, activating the functionalized polystyrene/divinylbenzene extraction column by using methanol and water in sequence, and discarding a collecting solution;
s4, taking the blank plasma matrix crude extract prepared in the step S2, loading the blank plasma matrix crude extract into the functionalized polystyrene/divinylbenzene extraction column activated in the step S3, and collecting effluent liquid to obtain blank plasma primary liquid;
s5, further placing the blank plasma matrix primary liquid prepared in the step S4 in a 3KD ultrafiltration tube, centrifuging for 15-20min at 10000-14000rpm, and discarding the filtrate; adding 300 plus 400 mu L of ultrapure water into the supernatant in an ultrafiltration tube, uniformly mixing, and centrifuging again at 10000 plus 14000rpm for 15-20 min; the filtrate was discarded and the above steps were repeated several times, after which the initial volume was made up with ultrapure water.
6. A blank plasma matrix obtained by the method according to any one of claims 1 to 5.
7. Use of a blank plasma matrix prepared according to the method of any one of claims 1-6 for target detection of a plasma sample.
8. Use according to claim 7, characterized in that: the target comprises one or more than one of adrenaline E, noradrenaline NE, methoxy adrenaline MN, dopamine DA, methoxy noradrenaline NMN and 3-methoxy tyramine 3-MT.
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|---|---|---|---|---|
| CN112014186A (en) * | 2020-09-07 | 2020-12-01 | 深圳华大临床检验中心 | Preparation method and application of blank blood matrix |
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| CN113820437B (en) | 2023-08-11 |
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