CN112014186B - Preparation method and application of blank blood matrix - Google Patents
Preparation method and application of blank blood matrix Download PDFInfo
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- CN112014186B CN112014186B CN202010927571.5A CN202010927571A CN112014186B CN 112014186 B CN112014186 B CN 112014186B CN 202010927571 A CN202010927571 A CN 202010927571A CN 112014186 B CN112014186 B CN 112014186B
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- 210000004369 blood Anatomy 0.000 title claims abstract description 54
- 239000008280 blood Substances 0.000 title claims abstract description 54
- 239000011159 matrix material Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 19
- 238000001179 sorption measurement Methods 0.000 claims abstract description 60
- 239000000523 sample Substances 0.000 claims abstract description 41
- 239000003463 adsorbent Substances 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 229940088594 vitamin Drugs 0.000 claims abstract description 16
- 229930003231 vitamin Natural products 0.000 claims abstract description 16
- 235000013343 vitamin Nutrition 0.000 claims abstract description 16
- 239000011782 vitamin Substances 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims abstract description 15
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- 229940088597 hormone Drugs 0.000 claims abstract description 11
- 239000005556 hormone Substances 0.000 claims abstract description 11
- 239000003480 eluent Substances 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 239000002858 neurotransmitter agent Substances 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 239000013595 supernatant sample Substances 0.000 claims abstract description 3
- 210000002966 serum Anatomy 0.000 claims description 33
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- 230000000694 effects Effects 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 14
- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 claims description 9
- 229910052901 montmorillonite Inorganic materials 0.000 claims description 9
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 210000002381 plasma Anatomy 0.000 claims description 2
- 230000003064 anti-oxidating effect Effects 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 description 11
- 239000003085 diluting agent Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 229930003571 Vitamin B5 Natural products 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 4
- 229960002079 calcium pantothenate Drugs 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 239000011675 vitamin B5 Substances 0.000 description 4
- 235000009492 vitamin B5 Nutrition 0.000 description 4
- 229930003451 Vitamin B1 Natural products 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229960003495 thiamine Drugs 0.000 description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 3
- 235000010374 vitamin B1 Nutrition 0.000 description 3
- 239000011691 vitamin B1 Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 235000019156 vitamin B Nutrition 0.000 description 2
- 239000011720 vitamin B Substances 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 229930003471 Vitamin B2 Natural products 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DHSUYTOATWAVLW-WFVMDLQDSA-N cilastatin Chemical group CC1(C)C[C@@H]1C(=O)N\C(=C/CCCCSC[C@H](N)C(O)=O)C(O)=O DHSUYTOATWAVLW-WFVMDLQDSA-N 0.000 description 1
- 229960004912 cilastatin Drugs 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940026231 erythorbate Drugs 0.000 description 1
- 235000010350 erythorbic acid Nutrition 0.000 description 1
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 description 1
- 229960004884 fluconazole Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 1
- 229960003907 linezolid Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- 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
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a preparation method and application of a blank blood matrix. The invention provides a method for preparing a blank blood matrix, which comprises the following steps: taking a blood sample to be tested, adjusting the pH to an optimal adsorption pH range, adsorbing the blood sample by using an adsorbent, and centrifugally separating the adsorbent from the blood sample; transferring the supernatant sample obtained after centrifugation into an ultrafiltration tube, and adding eluent for ultrafiltration elution; and centrifuging and collecting the supernatant to obtain the blank blood matrix. The blank blood matrix preparation method can be used for the preparation, production and application of the existing water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones and other detection systems, platforms, detection devices or hollow white samples in kits.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of a blank blood matrix.
Background
The blank blood matrix has great significance in clinical detection, has similar biological characteristics to a blood sample to be detected, does not contain a target detection object, and is commonly used as a blank control experiment sample or a blank diluent and the like. In the detection of vitamins, amino acids, neurotransmitters, drugs, hormones, etc., blank blood matrices are often used to formulate standard curves, quality control substances, etc. detection reagents.
The content of the residual substances to be measured in the blank blood matrix directly influences the accuracy of reagent configuration, so that the removal degree of the substances to be measured in the blank blood matrix is important. The adsorption material is used for adsorbing and removing the to-be-detected object, the reserved matrix is a common preparation mode of a blank blood matrix sample, but the adsorption efficiency of the adsorbent is influenced by various factors, and because of the difference of physical properties to be detected, the phenomenon of low competitive adsorption or effective adsorption rate exists, such as substances of vitamin B5, pyridoxine acid (VB 6 PA), amino acid and the like are difficult to remove through adsorption.
The residual problem of the blank serum/plasma matrix purchased in the market at present is common, wherein the water-soluble vitamin B1 is the most serious, and the detection and quantification are seriously affected.
Disclosure of Invention
The invention aims to provide a method for removing substances to be detected from a blood sample more comprehensively and thoroughly and obtaining a cleaner blank blood matrix sample.
In a first aspect, the invention claims a method of preparing a blank blood matrix.
The method for preparing the blank blood matrix as claimed in the invention can comprise the following steps:
(A1) Taking an in-vitro blood sample to be tested, adjusting the pH to an optimal adsorption pH range, adsorbing the blood sample by using an adsorbent, and centrifugally separating the adsorbent from the blood sample;
(A2) Transferring the supernatant sample obtained after the adsorption and centrifugation in the step (A1) into an ultrafiltration tube, and adding eluent to carry out ultrafiltration elution; centrifuging and collecting the upper eluted liquid to obtain the blank blood matrix.
Wherein the blank blood matrix is substantially free of water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones, etc.
In the method, the blood sample may be whole blood or serum or plasma.
In the method, the optimal adsorption pH range may be pH3.0-6.0.
Further, the optimal adsorption pH range is pH4.5-5.0.
In the method, the adsorbent is an adsorbent material having a high specific surface area.
Further, the adsorbent may be at least one of montmorillonite and activated carbon.
In the method, the eluent is an aqueous solution or physiological saline solution having an antioxidant effect or a reducing effect.
Further, the eluent may be at least one of dithiothreitol, ascorbic acid, erythorbate, an aqueous solution of disodium edetate, or a physiological saline solution.
In the method, the number of times of adsorption may be one or more times (e.g., two times). The ultrafiltration elution may be performed one or more times (e.g., four or five times).
In a specific embodiment of the present invention, in step (A1), the centrifugation is for 10min at 5000 g. In the step (A2), the centrifugation is carried out for 30min at 3000-4000 g.
When the adsorbent is montmorillonite, the pH is adjusted to 4.5. For the first time of the adsorption, the ratio of the sample blood to montmorillonite may be 50mL:800mg. If two or more adsorptions are required, the supernatant obtained by centrifuging after the previous adsorptions is added to the same amount of montmorillonite as the first adsorptions from the second adsorptions. And (3) during the adsorption, vibrating at a constant speed for 30min, standing for 30min, and centrifuging at 5000g for 10min to remove montmorillonite.
When the adsorbent is activated carbon, the pH is adjusted to 5.0. When the adsorption is carried out for the first time, the ratio of the blood sample to be tested to the activated carbon can be 50mL:50mg. If two or more adsorptions are required, the supernatant obtained by centrifuging after the previous adsorptions is added to the same amount of activated carbon as the first adsorptions from the second adsorptions. And (3) vibrating at a constant speed for 30min during the adsorption, standing for 6h, and centrifuging at 5000g for 10min to remove the adsorbent.
In a specific embodiment of the present invention, in step (A2), the molecular weight cut-off of the ultrafiltration tube is 10KD.
In step (A2), the method further comprises the step of fixing the volume of the supernatant obtained after the last ultrafiltration elution centrifugation to the original volume of the blood sample to be tested in step (A1) by using the eluent.
In a second aspect, the invention claims a blank blood matrix prepared by the method described above.
In a third aspect, the invention claims the use of a blank blood matrix as described above for formulating a standard curve (as a diluent) or quality control when performing target detection on a blood sample.
Further, the target may be a vitamin, amino acid, neurotransmitter, drug, hormone, or the like.
In a specific embodiment of the invention, the vitamin is specifically VB1, VB2 or VB5. The medicine is cilastatin, fluconazole or linezolid.
In the present invention, the blank blood matrix is a blank serum matrix free of water-soluble substances.
The invention has the beneficial effects that:
the blank blood matrix preparation method of the invention, first, adsorption and ultrafiltration elution technology are combined, can remove more than 99% of the objects to be detected; secondly, keeping the original biological characteristics of the blood sample unchanged; thirdly, because the eluent contains antioxidant components, the blank blood matrix sample prepared by the invention has certain storage stability.
Therefore, the blank blood matrix preparation method can be used for the preparation, production and application of the existing water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones and other detection systems, platforms, detection devices or hollow white samples in kits.
Drawings
FIG. 1 is a flow chart of the preparation process of a blank blood matrix sample according to the present invention.
Fig. 2 is a standard graph of several water-soluble vitamins for a blank serum configuration prepared using the blank blood matrix preparation method of the present application in an example of the present invention.
FIG. 3 is a standard graph of several antimicrobial agents prepared using the blank serum prepared by the blank blood matrix preparation method of the present application in the examples of the present invention.
FIG. 4 is a graph showing comparison of vitamin, hormone, and amino acid signal intensities in untreated and treated serum according to the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation of a blank serum matrix free of Water-soluble substances
A flow chart of the preparation process of the blank blood matrix sample is shown in FIG. 1.
(1) Taking 50mL of serum sample to be treated, regulating pH to 4.5, adding 800mg of montmorillonite (high-purity montmorillonite of Ming Biochemical Co., ltd. > 95%) adsorbent, vibrating at constant speed for 30min, standing for 30min, centrifuging at 5000g for 10min to remove the adsorbent, transferring supernatant to another centrifuge tube with 800mg of montmorillonite, repeating the above operations, and performing secondary adsorption;
(2) Transferring the secondary adsorption supernatant to a 70mL ultrafiltration tube (Milticore, 10 KD), centrifuging for 30min at 3000g, taking out the discarded filtrate, adding 10mL of 5 mu M DTT aqueous solution into the supernatant, mixing uniformly, centrifuging for 30min at 3000g again, taking out the discarded filtrate, adding 10mL of 5 mu M DTT aqueous solution into the supernatant, repeating the above operation for 3 times, and transferring to a new ultrafiltration tube for ultrafiltration elution.
(3) The supernatant was eluted and the volume was set to 50mL with 5. Mu.M DTT aqueous solution and stored for use.
EXAMPLE 2 preparation of blank plasma matrix free of Water-soluble substances
A flow chart of the preparation process of the blank blood matrix sample is shown in FIG. 1.
(1) Taking 50mL of plasma sample to be treated, regulating pH to 5.0, adding 50mg of activated carbon adsorbent (Sigma), vibrating at constant speed for 30min, standing for 6h, centrifuging at 5000g for 10min to remove the adsorbent, transferring the supernatant to another centrifuge tube with 50mg of activated carbon adsorbent, repeating the above operations, and performing secondary adsorption;
(2) Transferring the secondary adsorption supernatant to a 70mL ultrafilter tube (Milticore, 10 KD), centrifuging 4000g for 30min, taking out the discarded filtrate, adding 10mL of 5 μm DTT aqueous solution into the supernatant, mixing well, and centrifuging 4000g again for 30min; the filtrate was removed and discarded, 10mL of 5. Mu.M aqueous DTT was added to the supernatant, and the above procedure was repeated 4 times, during which time the solution was transferred to a new ultrafiltration tube for ultrafiltration elution.
(3) The supernatant was eluted and the volume was set to 50mL with 5. Mu.M DTT aqueous solution and stored for use.
Example 3 comparison of adsorbent adsorption and Ultrafiltration elution Effect
And comparing the influence of the adsorption of the adsorbent and the ultrafiltration elution technology on the removal effect of the B vitamins.
15mL of serum to be treated is taken respectively, activated carbon is taken as an adsorbent, the adsorbent is added, the primary adsorption of the adsorbent and the secondary adsorption of the adsorbent are respectively carried out, the primary adsorption of the adsorbent is combined with the primary ultrafiltration elution, 3 blank serum samples are prepared, and the detection is carried out by using liquid chromatography-tandem mass spectrometry. The method comprises the following steps:
the adsorbent is subjected to primary adsorption: and (3) 15mL of serum to be treated, regulating the pH to 5.0, adding 15mg of active carbon (Sigma), vibrating at a constant speed for 30min, standing for 6h, centrifuging for 10min at 5000g, and taking the adsorption supernatant, and fixing the volume to 15mL by using a 5 mu M DTT aqueous solution to obtain a blank serum sample.
And (3) secondary adsorption of the adsorbent: and (3) 15mL of serum to be treated, regulating the pH value to 5.0, adding 15mg of active carbon (Sigma), vibrating at a constant speed for 30min, standing for 6h, centrifuging for 10min at 5000g, removing the adsorbent, transferring the supernatant to another centrifuge tube with 50mg of active carbon adsorbent, repeating the above operation, performing secondary adsorption, taking the secondary adsorption supernatant, and fixing the volume to 15mL by using 5 mu M DTT aqueous solution to obtain a blank serum sample.
Adsorption of the adsorbent once and ultrafiltration elution once: 15mL of serum to be treated, regulating the pH to 5.0, adding 15mg of active carbon (Sigma), vibrating at a constant speed for 30min, standing for 6h, centrifuging for 10min by 5000g, transferring the adsorbed supernatant to a 70mL ultrafilter tube (Milpore, 10 KD), centrifuging for 30min by 4000g, taking out the discarded filtrate, adding 10mL of 5 mu M DTT aqueous solution into the supernatant, uniformly mixing, centrifuging for 30min again by 4000g, pouring out the supernatant eluted, and fixing the volume to 15mL by using 5 mu M DTT aqueous solution to obtain a blank serum sample.
The removal rates of VB1, VB2, VB5 and VB6PA in the 3 blank serum samples were detected by using liquid chromatography-tandem mass spectrometry.
The results are shown in Table 1. Therefore, the adsorption of the adsorbent can better adsorb and remove the vitamin B1 and the vitamin B2, but the average adsorption and removal effect on the vitamin B5 is less than 10 percent, and the average adsorption and removal effect on the vitamin B6PA is less than 50 percent. The number of adsorption times is increased, and the multiple adsorption is not improved obviously. The adsorption of the adsorbent and ultrafiltration elution are combined, so that VB5 and VB6PA can be obviously removed, and the average removal effect is more than 99%.
TABLE 1 adsorption of adsorbents and comparison of ultrafiltration elution effects
Example 4 pH Range test
This example will investigate the effect of the adsorption pH environment on the adsorption removal effect. The average adsorption removal effect of the adsorbents was compared at pH 1.5,5.0 and 8.0. The specific method comprises the following steps:
taking a serum sample to be treated, respectively adjusting the pH to 1.5,5.0 and 8.0, preparing a blank serum sample according to the operation steps of the example 2, and detecting the B vitamins in the blank serum sample prepared under the condition of 3 pH by adopting a liquid chromatography-tandem mass spectrometry.
The results are shown in Table 2. It can be seen that the adsorption removal effect of vitamin B1, vitamin B3 and vitamin B5 is low when the adsorption ph=1.5. When the adsorption ph=8.0, the adsorption removal effect of vitamin B5 and vitamin B7 is also low. The adsorption pH has no obvious difference for indexes such as other vitamins, amino acids, hormones and the like. The comprehensive test comparison considers that the optimal absorption pH range is 3.0-6.0.
TABLE 2 comparison of adsorption removal Effect for adsorption environments at different pH
EXAMPLE 5 blank serum matrix prepared according to the invention as a standard Curve diluent
The blank serum matrix prepared in the embodiment 1 is used as standard yeast diluent, standard yeast preparation of common water-soluble vitamins and antibacterial drugs is carried out, liquid phase mass spectrum detection is carried out, the concentration is taken as an abscissa, the Ratio (Area Ratio) of the detected substance to the peak Area corresponding to the internal standard is taken as an ordinate, and a standard curve is drawn.
The results are shown in FIGS. 2 and 3. As can be seen from the figure, the blank serum prepared by the blank blood matrix preparation method is used as the standard yeast diluent, the standard yeast is prepared for quantitative detection, and the linear correlation coefficient R of vitamins and antibacterial drugs is obtained 2 All are more than or equal to 0.99. The blank serum matrix prepared by the invention is clean, and can be used as standard curve diluent of detection substances such as vitamins, amino acids, neurotransmitters, hormones, medicines and the like.
Example 6 comparison of untreated serum samples and blank serum samples after treatment of example 1
The differences in the intensity of the responses of vitamins, hormones and amino acids in the untreated serum samples and the serum samples after the treatment of example 1 were detected using a liquid chromatography tandem mass spectrometry method.
As shown in FIG. 4, the response intensity of the serum sample treated in example 1 can be reduced by 2 orders of magnitude compared with that of the untreated serum sample, which means that the treatment of the method can significantly remove substances such as vitamins, hormones, amino acids and the like in the sample to obtain a clean matrix sample. Meanwhile, the chromatographic retention time of each compound is consistent before and after the sample is treated, which proves that the matrix effect of the blank serum matrix sample prepared by the method is not obviously different from that of the untreated serum sample.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. A method of preparing a blank blood matrix comprising the steps of:
(A1) Taking a blood sample to be tested, adjusting the pH to an optimal adsorption pH range, adsorbing the blood sample by using an adsorbent, and centrifugally separating the adsorbent from the blood sample;
(A2) Transferring the supernatant sample obtained after the adsorption and centrifugation in the step (A1) into an ultrafiltration tube, adding eluent to carry out ultrafiltration and centrifugal elution, and pouring out the supernatant eluted liquid to obtain the blank blood matrix;
the eluent is an aqueous solution with an antioxidation or a reduction effect, or the eluent is a physiological saline solution with an antioxidation or a reduction effect.
2. The method according to claim 1, characterized in that: the blood sample is whole blood or serum or plasma.
3. The method according to claim 1, characterized in that: the optimal adsorption pH range is pH3.0-6.0.
4. The method according to claim 1, characterized in that: the adsorbent is an adsorbent material having a high specific surface area.
5. The method according to claim 1, characterized in that: the adsorbent is at least one of montmorillonite and activated carbon.
6. The method according to claim 1, characterized in that: the eluent is at least one of the following: an aqueous solution of dithiothreitol, an aqueous solution of disodium edetate, a physiological saline solution of dithiothreitol, and a physiological saline solution of disodium edetate.
7. The method according to any one of claims 1-6, wherein: the number of times of adsorption is one or more times.
8. The method according to any one of claims 1-6, wherein: the ultrafiltration elution times are one or more times.
9. A blank blood matrix prepared by the method of any one of claims 1-8.
10. Use of the blank blood matrix of claim 9 in formulating a standard curve or quality control for target detection of a blood sample;
the target is a vitamin, amino acid, neurotransmitter, drug or hormone.
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