CN111398440B - Method for detecting arachidonic acid in blood - Google Patents
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Abstract
The invention discloses a method for detecting arachidonic acid in blood, which utilizes ultrafiltration membrane filaments to assist in enriching unsaturated fatty acid arachidonic acid in a blood sample. The specific method comprises the following steps: immersing the processed ultrafiltration membrane filaments in a blood sample, standing at normal temperature, taking out and drying, adding a proper amount of derivatization reagent, and synchronously performing elution and derivatization reactions.
Description
Technical Field
The invention belongs to the technical field of detection of metabolites in organisms, and particularly relates to a method for detecting arachidonic acid in blood by using ultrafiltration membrane filaments to assist in enriching metabolites in blood samples, wherein the metabolites are arachidonic acid which is unsaturated fatty acid.
Background
With the development of metabonomics in various fields of medical biology, the chromatography-mass spectrometry technology has become one of the most common technical means in an omics analysis platform. For small molecule metabolites such as amino acids, polysaccharides, polyols, vitamins, hormones, amides, cholesterol, polyamines, fatty acids, organic acids, phosphates, nucleotides, polypeptides and the like in a biomass sample (such as urine, blood, saliva, cells and the like), the method has the advantages of strong resolution, high sensitivity, good reproducibility and high throughput for chromatography-mass spectrometry.
Arachidonic Acid (AA), all-cis-5, 8, 11, 14-eicosatetraenoic acid, which contains four carbon-carbon double bonds and one carbon-oxygen double bond in the structure, belongs to omega-6 long chain polyunsaturated fatty acid, and is widely distributed in animal kingdom. AA is an important substance for the development of human brain and optic nerve, and has important effects on improving intelligence and enhancing visual acuity. AA plays an important role as phospholipid-bound structural lipid in blood, liver, muscle and other organ systems. AA has a series of physiological activities of esterifying cholesterol, increasing blood vessel elasticity, reducing blood viscosity, regulating blood cell function and the like, and has important effects of preventing cardiovascular diseases, diabetes, tumors and the like. Highly pure AA is a direct precursor for synthesizing eicosanoid derivatives such as prostaglandins, thromboxanes and leukotrienes, and these bioactive substances have important regulation effects on the metabolism of lipoprotein, hemorheology, vascular elasticity, leukocyte function and platelet activation. AA belongs to essential fatty acids in infancy and AA deficiency can have a serious adverse effect on the development of human tissues and organs, especially brain and nervous system development. After the human body grows, the human body can be converted from the essential fatty acids linoleic acid and linolenic acid, so the human body belongs to semi-essential fatty acids. Therefore, the measurement of the concentration of arachidonic acid in blood is of great importance for research in the relevant field. However, arachidonic acid is always difficult to detect and analyze blood metabolites at present.
The metabolite target analysis is to carry out sample pretreatment on a certain or a plurality of special components, remove impurity interference and improve the sensitivity of metabolite target analysis and detection. No report about detection of metabolites in blood samples enriched by ultrafiltration membrane filaments in an auxiliary manner is found at present, and no application of the ultrafiltration membrane filaments to analysis and detection of metabolite target arachidonic acid is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting arachidonic acid in blood, which utilizes ultrafiltration membrane filaments to assist in enriching arachidonic acid which is unsaturated fatty acid in a blood sample and realizes the rapid and accurate detection of the arachidonic acid.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for detecting arachidonic acid in blood, comprising:
1) washing the ultrafiltration membrane filaments by using an organic solvent, drying at room temperature, immersing the ultrafiltration membrane filaments in a blood sample to be detected, standing for 30-60 min, taking out the ultrafiltration membrane filaments, and drying at room temperature to obtain the ultrafiltration membrane filaments to be detected enriched with the target analytes;
2) placing the ultrafiltration membrane filaments to be tested in a derivatization reagent, synchronously carrying out elution and derivatization reaction at 65-75 ℃ for 30-50 min, and centrifuging reaction liquid obtained after the reaction is finished to obtain supernatant;
3) detecting the supernatant obtained in the step 2) by adopting a gas chromatography-mass spectrometry combined method.
The ultrafiltration membrane filaments of the present invention are obtained from commercially available hollow fiber ultrafiltration membranes. Hollow fiber ultrafiltration membranes, also commonly referred to as hollow fiber membranes, ultrafiltration membrane filaments, and the like, are fibrous in shape and have a hollow interior. When the hollow fiber ultrafiltration membrane is used, a plurality of hollow fiber ultrafiltration membranes are often gathered into curtains or bundles, and the hollow fiber ultrafiltration membrane is suitable for MBR (membrane bioreactor) membranes or external pressure column type ultrafiltration membranes.
In the invention, the ultrafiltration membrane filaments have selective adsorption and enrichment effects on arachidonic acid, and the possible principle is presumed as follows: after the ultrafiltration membrane filaments are treated by organic solvents such as methanol or acetone, the hydroxyl groups on the surfaces of the ultrafiltration membrane filaments are exposed. The arachidonic acid has four double bonds and one carboxyl group, and can be bonded with the hydroxyl on the ultrafiltration membrane filaments, so that the adsorption and enrichment of the ultrafiltration membrane filaments on the arachidonic acid are realized. The filtering effect of the microporous structure of the ultrafiltration membrane silk can be utilized to remove the interference of macromolecular proteins and various metabolites with complex structures in blood on target analytes.
The invention is also suitable for the compound with alcoholic hydroxyl, and the compound with alcoholic hydroxyl can be bonded with the exposed groups on the surface of the ultrafiltration membrane silk after treatment, thereby realizing active adsorption and enrichment.
The derivatization reagent is used as a derivatization reagent and an eluent at the same time, so that the arachidonic acid can be eluted from the ultrafiltration membrane wire, and the derivatization can be simultaneously carried out on the arachidonic acid, thereby facilitating the subsequent gas phase analysis.
In one embodiment: the length of the ultrafiltration membrane filaments is 0.5-2.0 cm.
In one embodiment: the ultrafiltration membrane filaments are polyvinyl chloride ultrafiltration membrane filaments.
In one embodiment: the inner diameter of the polyvinyl chloride ultrafiltration membrane wire is 0.8-1.2 mm, the outer diameter of the polyvinyl chloride ultrafiltration membrane wire is 1.6-1.7 mm, and the aperture of the polyvinyl chloride ultrafiltration membrane wire is 0.01-0.03 mu m.
In one embodiment: in the step 1), the organic solvent is methanol or acetone.
In one embodiment: in the step 1), the cleaning mode is soaking cleaning or ultrasonic cleaning.
In one embodiment: the blood sample to be tested is from a hyperlipidemic subject.
In one embodiment: the test blood sample is derived from a subject suffering from hyperlipidemia or acute myocardial infarction. The subject is a human or an animal.
According to the method, arachidonic acid is easier to detect for the blood sample with high blood fat. For example, hyperlipidemia rats and acute myocardial infarction patients (mostly with hyperlipidemia) have high blood lipid content in blood, and arachidonic acid is easy to detect.
In one embodiment: the derivatizing agent is a silylating agent.
In one embodiment: the addition amount of the derivatization reagent is 0.1-1 mL.
In one embodiment: the derivatization reagent is N, O-bis (trimethylsilyl) trifluoroacetamide BSTFA or N-methyl-N-trimethylsilane trifluoroacetamide MSTFA.
In one embodiment: the detection conditions of the gas chromatography-mass spectrometry combined method are as follows: agilent 7890A GC/5977B MSD, equipped with an electron impact ion source (EI), an HP-5MS chromatographic column, a film thickness of 25-35 m x 0.2-0.3 mm i.d., 0.2-0.3 μm, and using helium as a carrier gas and maintaining the sameIn the range of 1.1 to 1.3 mL/min -1 The flow rate of (a); the MS transmission line heater is 278-282 ℃; the ion source temperature is 248-252 ℃; the temperature of the four-level bar is 238-242 ℃; EI voltage is 68-72 eV; the temperature of a GC-MS sample inlet is 248-252 ℃: the column oven temperature program was as follows: the initial temperature is 59-61 ℃ and kept for 1.9-2.1 min, and the temperature is 9-11 ℃ per min -1 Heating to 278-282 ℃ and keeping for 19.5-20.5 min. The sample is injected in a split mode, and the split ratio is 1: 90 to 110.
Compared with the background technology, the technical scheme has the following advantages:
according to the invention, the unsaturated fatty acid arachidonic acid in a blood sample is enriched in an auxiliary way by virtue of the ultrafiltration membrane filaments of the environment-friendly product, so that the concentration of the arachidonic acid is increased in a short time. Immersing the treated ultrafiltration membrane wire in a blood sample to be tested, standing and extracting at normal temperature, taking out and drying, adding a proper amount of derivatization reagent, synchronously carrying out elution and derivatization reaction, removing the interference of macromolecular protein and metabolites with various complex structures in the blood sample on a target analyte due to the filtration effect of the microporous structure of the ultrafiltration membrane wire and the special selectivity of membrane wire materials, completing the enrichment of key metabolite arachidonic acid in the blood sample, increasing the concentration of the metabolite arachidonic acid in an analysis detection sample in a short time, simultaneously saving the sample preparation time, centrifuging the reaction liquid, taking supernatant for direct sample injection and analyzing by means of a gas chromatography-mass spectrometer. The method is simple, efficient and environment-friendly, and solves the problem that the arachidonic acid is difficult to accurately detect and analyze.
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The invention is further illustrated by the following figures and examples.
FIG. 1 is a selected ion flow diagram of example 1 of the present invention, with selected ions being 73, 117, 369.3, respectively, illustrating that the GC/MS analysis conditions of example 1 can be used for detecting arachidonic acid.
FIG. 2 is one of graphs showing experimental results of example 3 of the present invention, in which the target analyte is serum of a rat with hyperlipidemia, the upper graph is a total ion flow graph of arachidonic acid in serum, and the middle graph and the lower graph are respectively a mass spectrum and a library standard mass spectrum of arachidonic acid in serum, which illustrate that the sample pretreatment and GC/MS analysis conditions of example 3 can be used for detecting arachidonic acid in serum of a rat with hyperlipidemia.
Fig. 3 is a second experimental result diagram of embodiment 3 of the present invention, in which a target analyte is serum of an acute myocardial infarction patient, and a total ion flow diagram obtained by enriching a polyvinyl chloride ultrafiltration membrane filament MF1, a total ion flow diagram obtained by enriching a polypropylene ultrafiltration membrane filament MF2, a total ion flow diagram obtained by enriching a polyvinylidene fluoride ultrafiltration membrane filament MF3, and a control group, that is, a total ion flow diagram of serum not processed by an ultrafiltration membrane filament, are respectively provided from top to bottom, which illustrates that the polyvinyl chloride ultrafiltration membrane filament has a selective extraction and enrichment effect on arachidonic acid in serum of an acute myocardial infarction patient.
Detailed Description
The present invention will be described in detail with reference to the following examples:
example 1
The instrument comprises the following steps: GCMS (Agilent 7890A GC/5977B MSD Agilent), analytical balance (model XS205, Mettler-Torilat, Germany), ultrapure water system (Ming & clear TM-D24UV, Merck Millipore, Germany), three-frequency numerical control ultrasonic cleaner (model KQ-500VDB, Kunshan ultrasonic Instrument Co., Ltd.), centrifuge (model LXJ-II, Shanghai medical Analyzer).
Reagents and reagents: arachidonic acid (batch No. 1001004417, Sigma Co.); n, O-bis (trimethylsilyl) trifluoroacetamide (N, O-bis (trimethylsilyl) trifluoroacetamide, BSTFA), lot 7807, Shanghai Crystal pure reagents, Inc.; N-methyl-N-trimethylsilane trifluoroacetamide (N-methyl-N (trimethylsilly) trifluoroacetamide, MSTFA), lot No. 19478, aladin Chemistry co.ltd; the solvents such as chloroform, methanol, cyclohexane and absolute ethyl alcohol are used for the test, which is provided by Xiong science corporation for analytical purification.
The specific experimental steps comprise the following steps:
(1) preparing an arachidonic acid standard solution: 1mg is precisely weighed and placed in a volumetric flask, and a stock solution of 0.1mg/mL is prepared by chloroform for later use.
(2) GC/MS analysis conditions: agilent 7890A GC/5977B MSD, Agilent, equipped with an electron impact ion source (EI), Agilent G4513 autosampler, chromatography workstation and NIST 2011 mass spectrometry database; HP-5MS column, 30m × 0.25mm i.d., 0.25 μm film thickness (HP, USA), using helium (99.999%) as carrier gas and held at 1.2mL min -1 The flow rate of (c). The MS transmission line heater is 280 ℃; the ion source temperature is 250 ℃; the temperature of the four-level bar is 240 ℃; the EI voltage was 70 eV. The temperature of a GC-MS injection port is 250 ℃: the column oven temperature program was as follows: initial temperature 60 deg.C for 2min, and temperature 10 deg.C/min -1 The temperature was raised to 280 ℃ and maintained for 20 min. The sample is injected in a split mode, and the split ratio is 1: 100.
(3) the experimental result of the qualitative detection of arachidonic acid: the retention time of the arachidonic acid silanization reaction product in the total ion flow diagram is 20.841min, and the selected ion flow diagram is shown in FIG. 1.
Example 2
1. Preparation of serum for hyperlipidemic rat:
(1) experimental animals and feed: 10 SD rats, male, SPF grade, weight of 110-120 g, animals purchased from Shanghai Si Laike laboratory animals, Inc., animal production license number SCXK (Shanghai) 2017-: in the institute of medicine animal laboratory in Xiamen City. Room temperature: temperature of 21-23 ℃, humidity: 53 to 65 percent. Rat high fat feeds were purchased from shanghai pulotang biotechnology limited, feed production licenses: lu Fei certificate (2014)04001, production lot number 20181025-.
(2) Modeling experiment of hyperlipidemic rat: 10 male SD rats of SPF level are adaptively fed for 4 days, and basal feed is selected to eat freely without limitation of drinking water. Fasting was started for 12h at 8 o' clock after day 4, and body weight was weighed the next morning. Feeding with high-fat feed, continuously feeding for 6w, observing body weight, liver quality and liver index, measuring each index of rat serum, and observing pathological condition to show that the model is successfully formed.
(3) Preparation of serum for hyperlipidemic rat:
and (3) at the end of 6w administration, fasting the hyperlipidemia model rat for 12h without water prohibition, weighing the body mass in the morning of the next day, using diethyl ether for general anesthesia, taking blood from the heart, collecting 6-10 mL of whole blood, standing for 2h, centrifuging for 15min at 4 ℃ of a low-temperature centrifuge and 3000r/min, separating serum, and storing in a refrigerator at-20 ℃ to be tested.
2. Acute myocardial infarction serum sample preparation:
(1) acute myocardial infarction serum source: whole blood and serum samples from acute myocardial infarction patients were provided by the cardiovascular disease hospital affiliated with xiamen university: the study subjects met various inclusion criteria. The study had received patient consent and written informed consent for passage through the hospital ethics committee.
(2) Acute myocardial infarction serum sample preparation: 4mL of upper limb venous blood can be extracted when the patient with acute myocardial infarction is admitted. Separating serum from the collected blood sample after low-temperature high-speed centrifugation, and freezing at-80 ℃ for later use.
Example 3
1. Instruments and reagents: the same as in example 1.
Ultrafiltration membrane filaments: PVC ultrafiltration membrane filaments MF1 (inner/outer diameter: 1/1.66 mm); polypropylene ultrafiltration membrane filament MF2 (inner/outer diameter: 0.4/0.6 mm); the polyvinylidene fluoride ultrafiltration membrane filaments MF3 (inner/outer diameter: 0.9/1.5mm) with pore diameters of 0.02 μm were obtained from Guangzhou Haideke filtration Membrane science and technology Co., Ltd, and the filaments were cut into short pieces or cut from curtain-like or bundle-like membranes for subsequent treatment.
2. The specific experimental steps comprise the following steps:
(1) preparing ultrafiltration membrane filaments: cutting commercial ultrafiltration membrane filaments made of different materials into 1cm long, soaking in methanol, and cleaning for 15min, or cleaning for 5-15 min by ultrasonic, then performing ultrasonic treatment for 15-30 min (240W, 85Hz, 60 ℃), drying at room temperature, immersing in the serum of the rat with hyperlipidemia prepared in example 2, standing for extraction for 30-60 min, taking out, and drying at room temperature to obtain the ultrafiltration membrane filaments to be detected, which are enriched with the target analytes; placing the ultrafiltration membrane filaments to be detected in a dry 1.5mL centrifuge tube, adding 1mL BSTFA derivatization reagent, carrying out elution and derivatization reaction for 40min at 70 ℃, centrifuging the reaction solution obtained after the reaction is finished, and directly introducing the obtained supernatant into a gas chromatography-mass spectrometer for detection:
(2) GC/MS analysis: agilent 7890A GC/5977B MSD, Agilent, equipped with an electron impact ion source (EI), Agilent G4513 autosampler, chromatography workstation and NIST 2011 mass spectrometry database; HP-5MS chromatographic column, 30m in0.25mm i.d., 0.25 μm film thickness (HP, USA), using helium (99.999%) as carrier gas and held at 1.2mL min -1 The flow rate of (c). The MS transmission line heater is 280 ℃; the ion source temperature is 250 ℃; the temperature of the four-level bar is 240 ℃; the EI voltage was 70 eV. The temperature of a GC-MS injection port is 250 ℃: the column oven temperature program was as follows: initial temperature 60 deg.C for 2min, and temperature 10 deg.C/min -1 The temperature was raised to 280 ℃ and maintained for 20 min. The sample is injected in a split mode, and the split ratio is 1: 100.
(3) the result of the selection experiment of the ultrafiltration membrane silk film-forming material is as follows: after parallel comparative experiments, the polyvinyl chloride is proved to have selective extraction and enrichment effects on arachidonic acid, and the comparative analysis result is shown in figure 3.
(4) The analysis result of the polyvinyl chloride ultrafiltration membrane wire on the actual sample is as follows: according to the detection method, arachidonic acid (total ion flow diagram shown in figure 3) in the serum of a rat with hyperlipidemia (total ion flow diagram and mass spectrogram shown in figure 2) and an acute myocardial infarction patient can be effectively detected.
(5) The analysis results of the polyvinyl chloride ultrafiltration membrane filaments with different lengths on the same sample are as follows: the extraction experiments were carried out according to example 2, with the use of 1mL of derivatizing reagent, and the experimental analysis results showed that the content of enriched arachidonic acid gradually increased with the increase in length of the ultrafiltration membrane filaments, and therefore, the length of the ultrafiltration membrane filaments could be determined according to the difficulty of sample collection.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (7)
1. A method for detecting arachidonic acid in blood, which is characterized by comprising the following steps: the method comprises the following steps:
1) washing the ultrafiltration membrane filaments by using an organic solvent, drying at room temperature, immersing the ultrafiltration membrane filaments in a blood sample to be detected, standing for 30-60 min, taking out the ultrafiltration membrane filaments, and drying at room temperature to obtain the ultrafiltration membrane filaments to be detected enriched with the target analytes; the ultrafiltration membrane filaments are polyvinyl chloride ultrafiltration membrane filaments; the organic solvent is methanol or acetone;
2) placing the ultrafiltration membrane filaments to be tested in a derivatization reagent, synchronously carrying out elution and derivatization reaction at 65-75 ℃ for 30-50 min, and centrifuging reaction liquid obtained after the reaction is finished to obtain supernatant; the derivatization reagent is N, O-bis (trimethylsilyl) trifluoroacetamide BSTFA or N-methyl-N-trimethylsilane trifluoroacetamide MSTFA;
3) detecting the supernatant obtained in the step 2) by adopting a gas chromatography-mass spectrometry combination method; the detection conditions of the gas chromatography-mass spectrometry combined method are as follows: agilent 7890A GC/5977B MSD, equipped with an electron impact ion source, an HP-5MS chromatographic column, a film thickness of 25-35 m × 0.2-0.3 mm i.d., 0.2-0.3 μm, and using helium as carrier gas and keeping the helium at 1.1-1.3 mL/min -1 The flow rate of (a); the MS transmission line heater is 278-282 ℃; the ion source temperature is 248-252 ℃; the temperature of the four-level bar is 238-242 ℃; EI voltage is 68-72 eV; the temperature of a GC-MS sample inlet is 248-252 ℃; the column oven temperature program was as follows: the initial temperature is 59-61 ℃ and kept for 1.9-2.1 min, and the temperature is 9-11 ℃ per min -1 Heating to 278-282 ℃ and keeping for 19.5-20.5 min; the sample is injected in a split mode, and the split ratio is 1: 90 to 110.
2. The method according to claim 1, wherein: the length of the ultrafiltration membrane filaments is 0.5-2.0 cm.
3. The method according to claim 1, wherein: the inner diameter of the polyvinyl chloride ultrafiltration membrane wire is 0.8-1.2 mm, the outer diameter of the polyvinyl chloride ultrafiltration membrane wire is 1.6-1.7 mm, and the aperture of the polyvinyl chloride ultrafiltration membrane wire is 0.01-0.03 mu m.
4. The method according to claim 1, wherein: in the step 1), the cleaning mode is soaking cleaning or ultrasonic cleaning.
5. The method according to claim 1, wherein: the blood sample to be tested is from a hyperlipidemic subject.
6. The method according to claim 1, wherein: the test blood sample is derived from a subject suffering from hyperlipidemia or acute myocardial infarction.
7. The method according to claim 1, wherein: the addition amount of the derivatization reagent is 0.1-1 mL.
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| CA2445316A1 (en) * | 2001-04-26 | 2002-11-07 | Varian, Inc. | Hollow fiber membrane sample preparation devices |
| CN104073527A (en) * | 2014-06-16 | 2014-10-01 | 南京泽朗医药科技有限公司 | Method for preparing arachidonic acid and application of arachidonic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2445316A1 (en) * | 2001-04-26 | 2002-11-07 | Varian, Inc. | Hollow fiber membrane sample preparation devices |
| CN104073527A (en) * | 2014-06-16 | 2014-10-01 | 南京泽朗医药科技有限公司 | Method for preparing arachidonic acid and application of arachidonic acid |
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