CN114113426B - Method for detecting phospholipid in hemoglobin oxygen carrier - Google Patents
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- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 54
- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 17
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 17
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 239000001301 oxygen Substances 0.000 title claims abstract description 12
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims description 27
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 24
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 claims description 22
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims description 20
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims description 20
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 19
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 19
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 17
- 239000000126 substance Substances 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000001212 derivatisation Methods 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000007127 saponification reaction Methods 0.000 claims description 11
- 230000007062 hydrolysis Effects 0.000 claims description 10
- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000000413 hydrolysate Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Substances [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- WCTAGTRAWPDFQO-UHFFFAOYSA-K trisodium;hydrogen carbonate;carbonate Chemical compound [Na+].[Na+].[Na+].OC([O-])=O.[O-]C([O-])=O WCTAGTRAWPDFQO-UHFFFAOYSA-K 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 239000002585 base Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000003633 blood substitute Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000004305 normal phase HPLC Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 230000004075 alteration Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
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- 150000003384 small molecules Chemical class 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000003617 erythrocyte membrane Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- -1 polytetrafluoroethylene Polymers 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/89—Inverse chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
-
- 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
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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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)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of phospholipid detection, in particular to a detection method of phospholipid in a hemoglobin oxygen carrier. The invention eliminates a large amount of organic solvents which are not friendly to the environment when the normal phase chromatograph is used for measuring the phospholipid, solves the problems of lack of chromophores and low sensitivity of the normal phase chromatograph, and can be used for routine projects of the phospholipid in the hemoglobin oxygen carrier.
Description
Technical Field
The invention relates to the technical field of phospholipid detection, in particular to a detection method of phospholipid in a hemoglobin oxygen carrier.
Background
Phospholipids have a major impact in human health and in the development of disease formation. Accurate and efficient determination of phospholipids in vivo is helpful for understanding the metabolism of phospholipids in vivo and their role in life activities, thereby effectively diagnosing and preventing diseases.
The hemoglobin oxygen carrier is a blood substitute prepared by taking animal or human red blood cells as raw materials and adopting polymerization, crosslinking, coupling and other modes to chemically modify the separated and purified hemoglobin. Erythrocyte membrane phospholipids are mainly Phosphatidylethanolamine (PE) and Phosphatidylserine (PS). In the process of hemoglobin purification, the hemoglobin release causes the purified hemoglobin product to also generally contain phospholipids, which have significant toxicity and cause blood coagulation, while PS is an activator of prothrombin and promotes erythrocyte aggregation and adhesion to vascular endothelial cells to form thrombus, so the contents of PE and PS must be controlled.
The method for detecting phospholipids mainly comprises normal phase High Performance Liquid Chromatography (HPLC). Normal phase high performance liquid chromatography (NPLC) can achieve better separation between phospholipid classes, and the weak polar is eluted first, but the mobile phase often used in NPLC is weak polar, and bubbles are often generated when the mobile phase is mixed with a relatively strong polar solution, so that the problems of drift of retention time and uneven baseline are caused, and the use of a large amount of organic flow is relatively environment-friendly. Because of the lack of chromophores in the phospholipid molecular structure, detection is usually carried out in the range of 200-214nm, the absorbance coefficient of phospholipids in the wavelength range is small, the detection sensitivity is low, and some common solvents generally absorb light in the wavelength range strongly, so that detection of phospholipids is interfered. Mass Spectrometry (MS) is the most sensitive and specific detection technique that, in combination with HPLC, allows simultaneous separation, characterization and quantification of phospholipids, a disadvantage of common use in phospholipid spectroscopy is that detection is relatively expensive and unsuitable for routine analysis.
Disclosure of Invention
The invention aims to solve the technical problems and provide a method for detecting phospholipid in a hemoglobin-based oxygen carrier.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for detecting phospholipids in a hemoglobin-based oxygen carrier, comprising the steps of:
1) Extracting phospholipids to be detected from a test sample, wherein the phospholipids comprise phosphatidylethanolamine and phosphatidylserine;
2) Performing saponification hydrolysis on phospholipids in a sample to be tested by using a strong alkali solution to obtain hydrolysis products, wherein the hydrolysis products comprise ethanolamine and serine;
3) Introducing a chromophore into the molecular structure of the hydrolysate through the derivatization reaction of the tosyl chloride solution and the hydrolysate to obtain a sample derivatization product, passing through a water-based filter membrane, and detecting after chromatographic sample introduction;
4) And 2) processing the phospholipid standard substance solution in the steps 2) -3) to obtain standard substance derivative products, detecting the standard substance derivative products and the sample substance derivative products at 240nm by using reverse phase chromatography, plotting chromatographic peak areas and correlating the amounts of the phospholipids in the phospholipid standard substance solution to obtain a working curve equation, and substituting the peak areas of the sample substance derivative products into the working curve equation to obtain the amounts of different phospholipids in the sample to be tested.
Further, in S1, extracting the phospholipid to be detected specifically includes:
11 Adding equal volume of chloroform into the sample, shaking and mixing uniformly, adding 2 times of methanol into the sample, shaking and mixing uniformly, continuously adding chloroform and normal saline which are equal to the sample in volume, shaking and standing, layering, extracting the supernatant, and standing for later use;
12 Repeating the step 11) twice under the same conditions, combining the lower clear liquid, concentrating the lower clear liquid to 1/10 of the volume of the sample, and continuously drying to obtain the phospholipid to be detected.
In S2, the strong alkali is NaOH or KOH solution, the concentration of the strong alkali is 0.05-0.5 mol/L, and the volume of the strong alkali is 0.03-0.06 times of the volume of the sample.
Further, in S2, saponification and hydrolysis are carried out for 80-150 min at 70-90 ℃.
Further, in S2, after the saponification and hydrolysis are completed, HCl or H 2SO4 solution is used to neutralize excess strong base, and the concentration of the HCl or H 2SO4 solution is 0.5-3 mol/L.
Further, in S3, na 2CO3-NaHCO3 buffer solution with the concentration of 0.05-0.5 mol/L is added in the derivatization reaction, and the volume of the Na 2CO3-NaHCO3 buffer solution is 0.06-0.16 times of the volume of the sample.
Further, in S3, the volume of the p-toluenesulfonyl chloride solution is 0.02-0.1 times of the volume of the sample, and the concentration is 1.0-5.0 mg/ml, and the solution is prepared by acetonitrile solution.
Further, in S3, the reaction condition is 15-65 ℃ for 3-10 min.
Further, in S3, the pore size of the aqueous filter was 0.22 μm or 0.45. Mu.m.
The derivatization principle of the invention: after the phospholipid such as PE and PS is saponified and hydrolyzed, the molecular structures of the product ethanolamine and serine do not have chromophore groups, but the product ethanolamine and serine belong to primary amine, and are extremely easy to react with p-toluenesulfonyl chloride to generate Hisberg, and the chromophore groups are introduced into the structure, so that ultraviolet absorption detection is realized. The specific reaction principle is shown in figure 1. The maximum absorption wavelength of the derivative reaction product was 240nm, and the maximum absorption wavelength of p-toluenesulfonyl chloride was 230nm as shown in FIG. 2, so the detection wavelength was set to 240nm.
The invention has the beneficial effects that:
The method of the invention comprises the steps of saponifying the extracted phospholipids under alkaline conditions to generate small molecules (ethanolamine, serine and other), then chemically derivatizing the small molecules with p-toluenesulfonyl chloride, separating the derivative by reverse phase chromatography, and detecting the derivative at 240 nm. The method has the advantages that a large amount of organic solvents which are not friendly to the environment during the determination of phospholipid by normal phase chromatography are abandoned, a conventional reverse phase liquid chromatography mode with high separation degree is used, the problems of lack of chromophores and low sensitivity of normal phase chromatography are solved, and the method can be used for conventional project detection in laboratories.
Drawings
FIG. 1 shows the reaction principle of ethanolamine, serine and p-toluenesulfonyl chloride.
FIG. 2 is an ultraviolet absorption spectrum of p-toluenesulfonyl chloride and derivatives.
Fig. 3 is a sample measurement chromatogram, in which, a: a blood substitute; b: pig whole blood; c: pig serum.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Instruments and reagents used in the examples
Instrument: agilent 1260 high performance liquid chromatograph (Agilent company, usa); milli-Q ultra-pure water machine (Millipore Co., U.S.A.); MTN-2800D nitrogen blower (Beijing Hua Ruibo, manufactured by Tech Co., ltd.); MS3 basic vortex mixer (IKA, germany). BT 125D electronic balances (Sartorius, germany); constant temperature water bath pot and the like
Reagent: acetonitrile, methanol (chromatographic purity, sigma, usa), KOH, concentrated sulfuric acid, chloroform (analytical purity, national pharmaceutical chemicals limited); ultrapure water (Milli-Q ultrapure water machine).
Standard substance: phosphatidylethanolamine, phosphatidylserine standard (sigma us).
Example 1
1) Sample information: pig whole blood, pig serum and blood substitutes (glutaraldehyde polymeric hemoglobin solution, protein concentration: 115g/L; pH: 7.45).
2) Phospholipid extraction: transferring 5ml of the sample into a 60ml separating funnel (polytetrafluoroethylene piston), slowly adding 5ml of chloroform into the sample, shaking while adding 10ml of methanol, shaking to uniformly mix, continuously adding 5ml of chloroform and 5ml of physiological saline into the separating funnel, shaking, standing, layering, and extracting the supernatant into a 50ml centrifuge tube. After the extraction is completed, adding 5ml of chloroform into the sample again, repeating the steps twice under the conditions, and merging the supernatants; when the supernatant was blown to about the remaining 0.5ml with a nitrogen blower, the supernatant was carefully transferred to a 2ml centrifuge tube (50 ml centrifuge tube was rinsed with a small amount of chloroform, and the rinse was transferred to a 2ml centrifuge tube, repeated twice), and blow-drying was continued for use.
3) Saponification: 150 μl of 1mol/L KOH solution was added to the centrifuge tube and reacted at 90deg.C for 100min, and shaking was noted during heating to allow full reaction. After saponification is completed, 1mol/L sulfuric acid is added to adjust neutrality.
4) And (3) derivatization: to the saponified solution was added 700. Mu.l of 0.1mol/L Na 2CO3-NaHCO3 (pH=9.0) buffer, and the pH=9.0 was adjusted. 150 μl of 5.0mg/ml of p-toluenesulfonyl chloride was added and the derivatization was carried out at 60℃for 5 minutes, with shaking during heating, to ensure sufficient reaction. After the reaction is finished, the mixture is filtered through a water-based filter membrane with the thickness of 0.22 mu m, and sample injection is carried out.
5) Liquid chromatography conditions: chromatographic column: inertsil ODS-3 (5 μm, 4.6X1250 mm); mobile phase a:10mM ammonium acetate (ph=4.00); mobile phase B: chromatographic purity acetonitrile; elution gradient: isocratic elution in 19% b; chromatographic column temperature: 30 ℃; detection wavelength: 240nm; flow rate: 1.0ml/min; run time: 20min; sample injection amount: 20 μl.
To verify the effect of the method of the invention, the following observations were made:
1. investigation of the Linear relationship
The method verification is performed by measuring the following parameters: linearity, precision, accuracy, detection limit and quantification limit. The method uses an external standard method to quantify, and the concentration of the phospholipid standard substance is plotted against the chromatographic peak area after saponification and derivatization to obtain a working curve equation. The relative standard deviation RSD of the sample parallel assay to evaluate the precision of the method; the labeled recovery of the sample was used to evaluate the accuracy of the method;
The method uses an external standard method to quantify, and the concentration of the phospholipid standard substance is plotted against the chromatographic peak area after saponification and derivatization to obtain a working curve equation. The linearity of the method is shown in Table 1, the linearity range is 0.5-50 mug/mL, a good linear relation exists between the phospholipid concentration and the target peak area, and the correlation coefficient R 2 is 0.9927 (PS) and 0.9995 (PE) respectively.
The detection limit and the quantification limit were evaluated with 3-fold and 10-fold signal-to-noise ratios, respectively. As a result, the detection limit of PS was 0.2. Mu.g/mL and the quantitative limit was 0.5. Mu.g/mL as shown in Table 1; PE was detected at a limit of 0.1. Mu.g/mL, and the amount was determined at a limit of 0.3. Mu.g/mL.
TABLE 1 calibration Curve, detection limit and quantitative limit
2. Repetition and precision investigation
For the precision of the test method, pig serum samples were taken, the PS and PE contents were determined under optimized experimental conditions and repeated six times, and the data and results of the determination are shown in table 2. The RSD of the 6 parallel measurement is 3.7% and 4.4%, respectively, which shows that the precision of the method meets the requirement.
Table 2 repeatability test results (n=6)
3. Recovery rate and detection limit investigation
The method was used to determine PS and PE in porcine whole blood, porcine serum and blood substitutes, and the results are shown in FIG. 3 and Table 3. As can be seen from FIG. 3, the method has stable baseline and symmetrical target peak-to-peak type in chromatographic separation, basically achieves baseline separation, and is suitable for analyzing and measuring PS and PE in blood samples. Wherein the PS and PE contents in the whole pig blood are 258.6 mug/mL and 466.7 mug/mL respectively; the PS and PE contents in the pig serum are 14.1 mug/mL and 31.2 mug/mL respectively; PS and PE were not detected in the blood substitutes. The sample standard recovery rate of different levels is between 76.0% and 89.4%, which shows that the accuracy of the method meets the requirements.
TABLE 3 recovery and sensitivity test results
a Initial concentration of phospholipids in the raw sample.
In summary, in the invention, the phospholipid in the sample is primarily separated from some hydrophilic impurities in the sample through liquid-liquid extraction, and the phospholipid and some non-saponified hydrophobic components can be separated in the saponification process, so that the matrix interference in the sample is greatly reduced after the pre-separation is carried out twice, thereby improving the sensitivity and accuracy of the method. The verification result shows that the method can be used for accurately measuring PS and PE in blood samples
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (4)
1. The quantitative detection method of phospholipid in hemoglobin-based oxygen carrier is characterized by comprising the following steps:
1) Extracting phospholipids to be detected from a test sample, wherein the phospholipids comprise phosphatidylethanolamine and phosphatidylserine; the extracting of the phospholipid to be detected comprises the following steps:
11 Adding equal volume of chloroform into the sample, shaking and mixing uniformly, adding 2 times of methanol into the sample, shaking and mixing uniformly, continuously adding chloroform and normal saline which are equal to the sample in volume, shaking and standing, layering, extracting the supernatant, and standing for later use;
12 Repeating the supernatant liquid twice under the same conditions as in the step 11), combining the lower clear liquid, concentrating the lower clear liquid to 1/10 of the volume of the sample, and continuously drying to obtain the phospholipid to be detected;
2) Performing saponification hydrolysis on phospholipids in a sample to be tested by using a strong alkali solution to obtain hydrolysis products, wherein the hydrolysis products comprise ethanolamine and serine; the strong alkali is NaOH or KOH solution, the concentration of the strong alkali is 0.05-0.5 mol/L, and the volume of the strong alkali is 0.03-0.06 times of the volume of the sample; saponification hydrolysis is carried out for 80-150 min at 70-90 ℃;
3) Introducing a chromophore into the molecular structure of the hydrolysate through the derivatization reaction of the tosyl chloride solution and the hydrolysate to obtain a sample derivatization product, passing through a water-based filter membrane, and detecting after chromatographic sample introduction; the volume of the p-toluenesulfonyl chloride solution is 0.02-0.1 times of the volume of the sample, and the concentration is 1.0-5.0 mg/ml, and the p-toluenesulfonyl chloride solution is prepared by acetonitrile solution; the derivatization reaction condition is 15-65 ℃ for 3-10 min;
4) Step 2) -3) processing the phospholipid standard substance solution to obtain a standard substance derivative product, detecting the standard substance derivative product and a sample substance derivative product at 240nm by using reverse phase chromatography, plotting the chromatographic peak area and correlating the amount of phospholipid in the phospholipid standard substance solution to obtain a working curve equation, and substituting the peak area of the sample substance derivative product into the working curve equation to obtain the amounts of different phospholipids in the sample to be tested;
Liquid chromatography conditions: chromatographic column: inertsil ODS-3; mobile phase a:10mM ammonium acetate; mobile phase B: chromatographic purity acetonitrile; elution gradient: isocratic elution in 19% b; chromatographic column temperature: 30 ℃; detection wavelength: 240nm; flow rate: 1.0ml/min; run time: 20min; sample injection amount: 20 μl;
The linear model of the phosphatidylserine is y=10.607+14.593x, y is the concentration of the phosphatidylserine, x is the chromatographic peak area of the phosphatidylserine, and the linear range is 0.5-50 mug/mL;
The linear model of the phosphatidylethanolamine is y=2.132+22.284 x, y is the concentration of the phosphatidylethanolamine, x is the chromatographic peak area of the phosphatidylethanolamine, and the linear range is 0.5-50 mug/mL.
2. The method for quantitatively detecting phospholipids in hemoglobin-based oxygen carrier of claim 1, wherein in step 2), after the saponification and hydrolysis are completed, the excess strong base is neutralized with HCl or H 2SO4 solution, and the concentration of HCl or H 2SO4 solution is 0.5 to 3mol/L.
3. The method for quantitatively detecting phospholipids in hemoglobin-based oxygen carrier of claim 2, wherein in step 3), the Na 2CO3-NaHCO3 buffer solution with concentration of 0.05-0.5 mol/L is added in the derivatization reaction, and the volume of the buffer solution is 0.06-0.16 times of the volume of the sample.
4. The method for quantitatively detecting phospholipid in a hemoglobin-based oxygen carrier of claim 3, wherein in the step 3), the pore size of the aqueous filter is 0.22 μm or 0.45. Mu.m.
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