CN112014186A - Preparation method and application of blank blood matrix - Google Patents
Preparation method and application of blank blood matrix Download PDFInfo
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- CN112014186A CN112014186A CN202010927571.5A CN202010927571A CN112014186A CN 112014186 A CN112014186 A CN 112014186A CN 202010927571 A CN202010927571 A CN 202010927571A CN 112014186 A CN112014186 A CN 112014186A
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- 210000004369 blood Anatomy 0.000 title claims abstract description 55
- 239000008280 blood Substances 0.000 title claims abstract description 55
- 239000011159 matrix material Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 238000001179 sorption measurement Methods 0.000 claims abstract description 59
- 239000000523 sample Substances 0.000 claims abstract description 44
- 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 25
- 239000006228 supernatant Substances 0.000 claims abstract description 24
- 238000010828 elution Methods 0.000 claims abstract description 16
- 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 14
- 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 9
- 229940079593 drug Drugs 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 8
- 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
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 24
- 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
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 3
- 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 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 2
- 229940026231 erythorbate Drugs 0.000 claims description 2
- 235000010350 erythorbic acid Nutrition 0.000 claims description 2
- 210000002381 plasma Anatomy 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims 1
- 230000003064 anti-oxidating effect Effects 0.000 claims 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims 1
- 239000012496 blank sample Substances 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- 239000007864 aqueous solution Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 5
- 239000000243 solution Substances 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
- 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
- 239000003085 diluting agent Substances 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
- 238000007865 diluting Methods 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
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000047 product Substances 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
- 230000006978 adaptation Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- DHSUYTOATWAVLW-WFVMDLQDSA-N cilastatin Chemical compound 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
- 238000010586 diagram Methods 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
- 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
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry 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
- 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
- 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
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- 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
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- 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 value to the 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 to an ultrafiltration tube, and adding an eluent for ultrafiltration elution; and centrifuging to collect the supernatant to obtain the blank blood matrix. The preparation method of the blank blood matrix can be used for preparing, producing and applying blank samples in the existing detection systems, platforms, detection devices or kits for water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones and the like.
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 important significance in clinical detection, and is commonly used as a blank control experiment sample or a blank diluent and the like because the blank blood matrix has biological characteristics similar to those of a blood sample to be detected and does not contain a target detection object. In the detection of vitamins, amino acids, neurotransmitters, drugs, hormones and the like, blank blood matrixes are often used for configuring detection reagents such as standard curves, quality control products and the like.
The content of the substance to be detected remaining in the blank blood matrix directly affects the accuracy of reagent preparation, so the removal degree of the substance to be detected in the blank blood matrix is very important. The substances to be detected are adsorbed and removed by using the adsorbing materials, the reserved matrix is a common preparation method of blank blood matrix samples, but the adsorption efficiency of the adsorbing agent is influenced by various factors, and due to the difference of physicochemical properties of the substances to be detected, the phenomenon of competitive adsorption or low effective adsorption rate exists, and substances such as vitamin B5, pyridoxine (VB6PA), amino acid and the like are difficult to remove by adsorption.
The blank serum/plasma matrix purchased in the market at present has more common residue problems, wherein the water-soluble vitamin B1 is the most serious, and the detection and the quantification are seriously influenced.
Disclosure of Invention
The invention aims to provide a method for more comprehensively and thoroughly removing substances to be detected from a blood sample to obtain a cleaner blank blood matrix sample.
In a first aspect, the invention claims a method of preparing a blank blood substrate.
The method for preparing blank blood matrix claimed by the invention can comprise the following steps:
(A1) taking an isolated blood sample to be tested, adjusting the pH value to the 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) to an ultrafiltration tube, and adding an eluent to carry out ultrafiltration elution; centrifuging to collect the upper layer eluate to obtain the blank blood matrix.
Wherein the blank blood matrix is substantially free of water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones and the like.
In the method, the blood sample may be whole blood or serum or plasma.
In the method, the optimum adsorption pH range may be pH 3.0-6.0.
Further, the optimum adsorption pH range is pH 4.5-5.0.
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 a physiological saline solution having an antioxidant effect or a reducing effect.
Further, the eluent may be at least one of dithiothreitol, ascorbic acid, erythorbate, disodium edetate in aqueous solution or physiological saline solution.
In the method, the number of times of adsorption may be one or more (e.g., two). The number of ultrafiltration elutions may be one or more (e.g., four or five).
In a specific embodiment of the present invention, in step (A1), the centrifugation is 5000g for 10 min. In the step (A2), the centrifugation is 3000-4000g for 30 min.
When the adsorbent is montmorillonite, the pH is adjusted to 4.5. When the adsorption is carried out for the first time, the ratio of the blood sample to be tested to the montmorillonite can be 50 mL: 800 mg. If necessary, carrying out two or more times of adsorption, and adding the supernatant obtained by centrifugation after the previous adsorption into the montmorillonite with the same amount as that of the first adsorption from the second adsorption. During the adsorption, shaking at 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 performed for the first time, the ratio of the blood sample to be tested to the activated carbon may be 50 mL: 50 mg. If the adsorption is needed to be carried out twice or more, the supernatant obtained by centrifuging after the previous adsorption is added into the activated carbon with the same quantity as the first adsorption from the second adsorption. And during the adsorption, shaking at a constant speed for 30min, 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 ultrafiltration tube has a molecular weight cut-off of 10 kD.
In the step (a2), the method further comprises the step of using the eluent to centrifuge the last ultrafiltration elution, and then adding the supernatant obtained after the last ultrafiltration elution to the original volume of the blood sample to be tested in the step (a 1).
In a second aspect, the invention claims a blank blood matrix prepared by the method described hereinbefore.
In a third aspect, the present invention claims the use of a blank blood matrix as described hereinbefore for formulating a standard curve (as a diluent) or a quality control for target detection of a blood sample.
Further, the target may be a vitamin, an amino acid, a neurotransmitter, a drug, a hormone, or the like.
In a particular embodiment of the invention, the vitamin is in particular VB1, VB2 or VB 5. The drug is specifically cilastatin, fluconazole or linezolid.
In the present invention, the blank blood substrate is a blank serum substrate containing no water-soluble substance.
The invention has the beneficial effects that:
the blank blood matrix preparation method of the invention, first, the absorption is combined with the ultrafiltration elution technology, more than 99% of the substance to be measured can be removed; secondly, keeping the original biological characteristics of the blood sample unchanged; thirdly, the blank blood matrix sample prepared by the invention has certain storage stability because the eluent contains antioxidant components.
Therefore, the blank blood matrix preparation method can be used for preparing, producing and applying blank samples in the existing detection systems, platforms, detection devices or kits for water-soluble vitamins, amino acids, neurotransmitters, drugs, hormones and the like.
Drawings
FIG. 1 is a flow chart of a process for preparing a blank blood matrix sample according to the present invention.
FIG. 2 is a standard graph of several water-soluble vitamins in a blank serum prepared by the blank blood matrix preparation method of the present application in an example of the present invention.
FIG. 3 is a standard curve diagram of several antibacterial agents configured with blank serum prepared by the blank blood matrix preparation method of the present application in the example of the present invention.
FIG. 4 is a comparison of the signal intensity of vitamins, hormones, amino acids in untreated and treated serum according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of a blank serum base without Water-soluble substances
The flow chart of the preparation process of the blank blood matrix sample of the present invention is shown in FIG. 1.
(1) Taking 50mL of a serum sample to be treated, adjusting the pH value to 4.5, adding 800mg of montmorillonite (high-purity montmorillonite of Chifeng and Mingsheng chemical engineering Co., Ltd. > 95%) adsorbent, shaking at a constant speed for 30min, standing for 30min, centrifuging for 10min at 5000g to remove the adsorbent, transferring the supernatant into another centrifugal tube with 800mg of montmorillonite, repeating the above operations, and performing secondary adsorption;
(2) transferring the supernatant of the second adsorption to a 70mL ultrafiltration tube (Milipore, 10KD), centrifuging for 30min at 3000g, taking out and discarding the filtrate, adding 10mL of 5 μ M aqueous DTT solution into the supernatant, mixing uniformly, centrifuging for 30min again at 3000g, taking out and discarding the filtrate, adding 10mL of 5 μ M aqueous DTT solution into the supernatant, repeating the above operation for 3 times, and transferring to a new ultrafiltration tube for ultrafiltration and elution.
(3) The supernatant was decanted off, and 50mL of the supernatant was made up with 5. mu.M aqueous DTT solution and stored for further use.
Example 2 preparation of blank plasma matrix without Water-soluble substances
The flow chart of the preparation process of the blank blood matrix sample of the present invention is shown in FIG. 1.
(1) Taking 50mL of a plasma sample to be treated, adjusting the pH value to 5.0, adding 50mg of activated carbon adsorbent (Sigma), shaking at a constant speed for 30min, standing for 6h, centrifuging 5000g for 10min to remove the adsorbent, transferring the supernatant into another centrifugal tube with 50mg of activated carbon adsorbent, repeating the above operations, and performing secondary adsorption;
(2) transferring the supernatant of the secondary adsorption to a 70mL ultrafiltration tube (Milipore, 10KD), centrifuging for 30min at 4000g, taking out and discarding the filtrate, adding 10mL of 5 mu M DTT aqueous solution into the supernatant, mixing uniformly, and centrifuging for 30min again at 4000 g; the filtrate was removed and discarded, and 10mL of 5. mu.M aqueous DTT solution was added to the supernatant, and the above operation was repeated 4 times, during which time the supernatant was transferred to a new ultrafiltration tube for ultrafiltration elution.
(3) The supernatant was decanted off, and 50mL of the supernatant was made up with 5. mu.M aqueous DTT solution and stored for further use.
Example 3 comparison of adsorbent adsorption and Ultrafiltration elution Effect
Comparing the influence of the adsorption of the adsorbent and the combination of the adsorbent and the ultrafiltration elution technology on the removal effect of the B vitamins.
Taking 15mL of serum to be treated, taking activated carbon as an adsorbent, adding the adsorbent, respectively carrying out primary adsorption on the adsorbent, carrying out secondary adsorption on the adsorbent, combining the primary adsorption on the adsorbent with primary ultrafiltration elution, preparing 3 parts of blank serum samples, and detecting by using liquid chromatography tandem mass spectrometry. The method comprises the following specific steps:
primary adsorption of an adsorbent: adjusting the pH value of 15mL of serum to be treated to 5.0, adding 15mg of activated carbon (Sigma), shaking at a constant speed for 30min, standing for 6h, centrifuging for 10min at 5000g, taking the adsorption supernatant, and diluting to 15mL with 5 mu M DTT aqueous solution to obtain a blank serum sample.
Secondary adsorption of an adsorbent: 15mL of serum to be treated, adjusting the pH value to 5.0, adding 15mg of activated carbon (Sigma), shaking at a constant speed for 30min, standing for 6h, centrifuging for 10min at 5000g, removing the adsorbent, transferring the supernatant into another centrifugal tube with 50mg of activated carbon adsorbent, repeating the above operations, performing secondary adsorption, taking the supernatant of the secondary adsorption, and diluting to 15mL with 5 mu M DTT aqueous solution to obtain a blank serum sample.
The adsorbent is subjected to one-time adsorption and one-time ultrafiltration elution: 15mL of serum to be treated, adjusting the pH value to 5.0, adding 15mg of activated carbon (Sigma), shaking at a constant speed for 30min, standing for 6h, centrifuging for 10min at 5000g, transferring the adsorption supernatant into a 70mL ultrafiltration tube (Milipore, 10KD), centrifuging for 30min at 4000g, taking out, discarding the filtrate, adding 10mL of 5 mu M DTT aqueous solution into the supernatant, mixing uniformly, centrifuging for 30min at 4000g again, pouring out the supernatant eluate, and diluting to 15mL with 5 mu M DTT aqueous solution to obtain a blank serum sample.
The removal rates of VB1, VB2, VB5 and VB6PA in the above 3 blank serum samples were examined using liquid chromatography tandem mass spectrometry.
The results are shown in Table 1. It can be seen that the adsorbent can adsorb and remove vitamin B1 and vitamin B2 well, but the average adsorption removal effect on vitamin B5 is less than 10%, and the average adsorption removal effect on vitamin B6PA is less than 50%. The adsorption times are increased, and the multiple adsorption is still not improved obviously. By combining the adsorption of the adsorbent with ultrafiltration elution, VB5 and VB6PA can be obviously removed, and the average removal effect reaches over 99 percent.
TABLE 1 comparison of adsorbent adsorption and Ultrafiltration elution Effect
Example 4 pH Range testing
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 values 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 values 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 was low when the adsorption pH was 1.5. The adsorption removal effect of vitamin B5 and vitamin B7 was also low when the adsorption pH was 8.0. And for indexes such as other vitamins, amino acids, hormones and the like, the adsorption pH has no obvious difference. The optimal adsorption pH range is 3.0-6.0 in comprehensive test and comparison.
TABLE 2 comparison of adsorption removal effect in different pH adsorption environments
Example 5 blank serum substrate prepared according to the invention as a dilution of standard
The blank serum base prepared in example 1 was used as a dilution of a standard curve, and a standard curve was prepared by preparing a common water-soluble vitamin and antibacterial drug standard curve and detecting the concentration by liquid phase mass spectrometry, and plotting the concentration as abscissa and the Ratio of peak areas of the detection substance to the corresponding internal standard (Area Ratio) as ordinate.
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 of the application is used as a standard curve diluent to prepare a standard curve for quantitative detection, and the linear correlation coefficient R of the vitamin and the antibacterial drug2Are all more than or equal to 0.99. The blank serum substrate prepared by the method 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 sample and blank serum sample after treatment in example 1
Differences in the intensity of vitamin, hormone and amino acid responses were detected in the untreated serum sample and the serum sample after treatment in example 1 using a liquid chromatography tandem mass spectrometry method.
As shown in FIG. 4, the response intensity of the serum sample treated in example 1 is reduced by 2 orders of magnitude on average compared with the response intensity of the untreated serum sample, which indicates that the treatment of the method of the present invention can significantly remove substances such as vitamins, hormones, and amino acids from the sample, and obtain a clean matrix sample. Meanwhile, the chromatographic retention time of each compound is consistent before and after the sample is treated, which shows that the matrix effect of the blank serum matrix sample prepared by the method has no obvious difference from that of the untreated serum sample.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.
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 value to the 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) to an ultrafiltration tube, and adding an eluent to carry out ultrafiltration elution; and centrifuging to collect the supernatant to obtain the blank blood matrix.
2. The method of claim 1, wherein: the blood sample is whole blood or serum or plasma.
3. The method according to claim 1 or 2, characterized in that: the optimum adsorption pH range is pH 3.0-6.0.
4. A method according to any one of claims 1-3, characterized in that: the adsorbent is an adsorbent material having a high specific surface area.
5. The method according to any one of claims 1-4, wherein: the adsorbent is at least one of montmorillonite and activated carbon.
6. The method according to any one of claims 1-5, wherein: the eluent is water solution or normal saline solution with antioxidation or reduction function.
7. The method according to any one of claims 1-6, wherein: the eluent is at least one of dithiothreitol, ascorbic acid, erythorbate, and disodium ethylene diamine tetraacetate water solution or normal saline solution.
8. The method according to any one of claims 1-7, wherein: the adsorption frequency is one or more than one time; and/or
The ultrafiltration elution is carried out once or more than once.
9. A blank blood substrate prepared by the method of any one of claims 1-8.
10. Use of the blank blood matrix of claim 9 for formulating a standard curve or quality control for target detection of a blood sample;
further, the target is a vitamin, an amino acid, a neurotransmitter, a drug, or a hormone.
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