CN111351881A - Method for detecting arachidonic acid and metabolite thereof in human plasma - Google Patents
Method for detecting arachidonic acid and metabolite thereof in human plasma Download PDFInfo
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Abstract
The invention aims to provide a method for detecting arachidonic acid in human plasma and metabolites thereof, which comprises the steps of adding isotope arachidonic acid into a biological sample as an internal standard, extracting arachidonic acid in a sample mixture by a liquid-liquid extraction method, separating the arachidonic acid in the sample by using a high-efficiency liquid phase, and performing tandem mass spectrometry by using an internal standard curve method, and has the advantages of good selectivity, high sensitivity, short analysis time, low mass spectrum signal-to-noise ratio and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for accurately and efficiently extracting and detecting pentadecanoic acid and metabolites thereof in human plasma.
Background
Arachidonic acid is an important substance for the development of human brain and optic nerve, and has important effects on improving intelligence, improving memory and enhancing visual acuity. Arachidonic acid also has a series of physiological activities of reducing cholesterol, increasing blood vessel elasticity, inhibiting platelet aggregation, reducing blood viscosity, regulating blood cell function and the like. Arachidonic acid has 3 metabolic pathways: (1) cyclooxygenase COX; (2) lipoxygenase LOX; (3) cytochrome P450 enzymes (CYP) are direct precursors for synthesizing icosic derivatives such as prostaglandins (prostaglandins), thromboxanes (thromboxanes) and leukotrienes (leukotrines), and these bioactive substances are involved in the regulation of inflammatory reaction and immune system, regulating vascular tension and platelet aggregation, regulating the growth of tumor cells, influencing the synthesis and release of hormones in endocrine system, and playing an important role in the development of central nervous system and the development of retinal nerve, so that the cytochrome P450 enzymes are related to various diseases such as atherosclerosis, hypertension, allergy, arthritis, and the like.
However, the existing pretreatment preparation method for the substances has the problems of high cost, long extraction time, low sample yield, difficulty in detection and the like. Therefore, how to develop a method which is low in cost, quick and capable of improving the yield of a target product, improving the detection limit of a mass spectrum and reducing detection noise is a key technical problem to be solved in the prior art.
Disclosure of Invention
The invention aims to provide a method for detecting arachidonic acid in human plasma and metabolites thereof, which comprises the steps of adding isotope arachidonic acid into a biological sample as an internal standard, extracting arachidonic acid in a sample mixture by a liquid-liquid extraction method, separating the arachidonic acid in the sample by using a high-efficiency liquid phase, and performing tandem mass spectrometry by using an internal standard curve method, and has the advantages of good selectivity, high sensitivity, short analysis time, low mass spectrum signal-to-noise ratio and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for detecting arachidonic acid and metabolites thereof in human plasma comprises the following steps:
step S1, dissolving an isotope internal standard product arachidonic acid by using methanol, preparing a mixed internal standard working solution, dissolving the standard product arachidonic acid by using a methanol aqueous solution, and preparing a standard product mixed solution;
step S2, using ethanol to dilute the standard substance mixed liquor obtained in the step S1 into a series of concentration standard substance mixed liquor with a plurality of concentration gradients in a gradient concentration manner, wherein the concentration gradients are more than three; respectively adding the mixed internal standard working solution in the step S1 and methanol containing 1% of FA into the mixed solution of the standard substance with each gradient concentration, carrying out ice bath ultrasonic treatment, centrifuging at low temperature, and then taking the supernatant to carry out liquid-liquid extraction to obtain an eluted filtrate after extraction for later use;
step S3, taking a sample to be detected, adding mixed internal standard working solution and methanol containing 1% of FA into the sample to be detected by the same operation method of step S2, carrying out ice bath ultrasonic treatment, centrifuging at low temperature, taking supernatant, and carrying out liquid-liquid extraction to obtain extracted filtrate for later use;
step S4, carrying out high performance liquid chromatography-tandem mass spectrometry on the extracted samples obtained in the steps S2 and S3, wherein the high performance liquid chromatography conditions comprise sample introduction at 4 ℃, column temperature of 35 ℃, flow rate of 400 mu L/min and sample introduction amount of 2 mu L; the liquid phase gradient is as follows, 0-1min, the B phase is maintained at 30%; 1-7min, the phase B changes linearly from 30% to 80%; 7-9min, phase B changes linearly from 80% to 90%; 9-11min, keeping phase B at 90%; 11-11.2min, phase B changes linearly from 90% to 30%; 11.2-14min, keeping phase B at 30%;
the mass spectrum detection is in a multi-reaction monitoring mode, and the ion source condition is that the ion source temperature (sourcetemperature) is 500 ℃; an atomization Gas pressure (ion Source Gas1) of 50; assist Gas pressure (Ion Source Gas 2): 50; air Curtain gas (curtaingas): 30, of a nitrogen-containing gas; spray Voltage (ionsafe Voltage flowing): 4500V.
According to the above protocol, the isotopic internal standard Arachidonic acid in step S comprises Thromboxane B-D (Thromboxane B-D), Leukotriene B-D (Leukotriene B-D), Leukotriene D-D (Leukotriene D-D), Prostaglandin F-4 (Prostagladin F-4), 8-isoprostane F0-D (8-iso-prostagladin F1-D), 6-keto-prostagladin F-D (6-keto-prostagladin F-D), 12 hydroxyeicosatetraenoic acid-D (12S-HETE-D), Arachidonic acid-D (Arachidonic acid-D), Prostaglandin D-D (Prostagladin D-D), (9S,10E,12Z) -9-hydroxyoctadecane-10, 12-dienoic acid-D (9S-DE-D), (9Z,11E) -13-hydroxy-9, 11-octadecane-D, 9S-12-hydroxyeicosatetraenoic acid-D (9S-D), Arachidonic acid-D (2E-12E, 12E-hydroxy octadecane-10, 12-10-octadecane-10, 12-dienoic acid-D (9S-D, 10E, 12-13-oxadocosatetraenoic acid) and 15-15% of each of a mixture of a working mixture solution, a mixture of Thromboxane-15-oxadocosatetraenoic acid (15-7-15-oxadiene-7-15 and 15-oxadiene-2-7-15-2-oxadiene-2-oxatrine mixture of a working mixture of a Thromboxane-oxadiene-oxatriene-2-7-2-oxatriene-7-15-one of a working mixture of a Thromboxane, a working mixture of a Thromboxane-oxatriene-2-oxatriene-2-one of a working mixture of a thrombodode-2-oxatriene-2-oxatriene-oxatrie.
According to the above scheme, the methanol containing 1% of FA in the steps S2 and S3 further contains an antioxidant; the ice bath ultrasonic treatment is 500W ultrasonic treatment in an ice bath for 30 min; the low-temperature centrifugation is performed for 10min at 4 ℃ at 14000 g.
According to the scheme, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the preparation method comprises the steps of weighing 2, 6-di-tert-butyl-p-cresol, adding methanol according to the proportion of 2.32mg/mL for dissolving, adding formic acid with the volume of 1/100 methanol, and uniformly mixing.
According to the above scheme, the liquid-liquid extraction in steps S2 and S3 is specifically implemented by using Oasis HLB solid phase extraction 96-well Plate (96-well Plate), adding 3mL of methanol activated well Plate in 3 times, each time adding 1 mL; adding 3mL of pure water balance pore plate for 3 times, wherein 1mL of pure water balance pore plate is added each time; adding the supernatant prepared in the step S2 or S3 into a pore plate; adding 2mL of cleaning solution A (washing solution A) in 2 times, and adding 1mL each time; adding 2mL of cleaning solution B (washing solution A) in 2 times, and adding 1mL of cleaning solution B each time; and finally, adding 1mL of methanol to elute the sample to obtain eluted filtrate.
According to the scheme, the cleaning solution A (washing solution A) is prepared by mixing the following components in a volume ratio of 19: 1-water: the antioxidant (2, 6-ditertbutyl-p-cresol solution) is mixed according to the proportion; the cleaning solution B (washing solution B) is prepared from the following components in a volume ratio of 10000: 1-cleaning liquid a: mixing with formic acid.
The invention has the beneficial effects that:
1) the sample is pretreated by liquid-liquid extraction, and the pretreatment of the sample is simple and quick.
2) The method has the advantages that the pretreatment step of the sample can be completed only by simple liquid-liquid extraction on the body fluid sample, 15 arachidonic acid can be effectively separated by using a Waters ACQUITY UPLC I-Class system, the quantification is carried out by 13 kinds of site internal standards, the important arachidonic acid and metabolites thereof in human plasma can be accurately detected, the method has higher precision and accuracy, can be used for quantitative analysis of various clinical samples, is simple in experimental operation and short in experimental period, provides a reliable detection method for the clinical health assessment of the arachidonic acid level, and provides an effective research means for scientific research.
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FIG. 1 is a diagram of a standard XIC of arachidonic acid and its metabolites in examples.
Fig. 2 is a graph of the DHA standard curve results in the examples.
The two drawings are display graphs of the result of the instrument analysis, only the result of the current experiment is displayed, the change can occur according to the difference of each actual experiment, and the characters in the drawings do not influence the repeated implementation of the patent scheme.
Detailed Description
The technical solution of the present invention is described below with reference to the accompanying drawings and examples.
A method for detecting arachidonic acid and metabolites thereof in human plasma comprises the following steps:
step S1, preparing a mixed internal standard working solution, namely dissolving an isotope internal standard product arachidonic acid comprising thromboxane B2-D4, leukotriene B4-D4, leukotriene D4-D5, prostaglandin F2 α -4, 8-isoprostaglandin F2 α -D4, 6-ketone-prostaglandin F1 α -D4, 12-hydroxyeicosatetraenoic acid-D8, arachidonic acid-D8, prostaglandin D2-D4, (9S,10E,12Z) -9-hydroxyoctadeca-10, 12-dienoic acid-D4, (9Z,11E) -13-hydroxy-9, 11-octadecadienoic acid-D4, 15-hydroxyeicosatetraenoic acid-D8 and docosahexaenoic acid-D5 by using methanol, and diluting the mixed internal standard working solution with water to obtain mixed solution of which the final concentration of each component in the mixed solution is 200 ng/ml;
preparing a standard substance mixed solution, namely dissolving various standard substances of arachidonic acid including thromboxane B2, leukotriene B4, leukotriene D4, prostaglandin E2, 8-isoprostane F2 α, prostaglandin F2 α, 6-keto-prostaglandin F1 α, prostaglandin D2, (9S,10E,12Z) -9-hydroxyoctadeca-10, 12-dienoic acid, (9Z,11E) -13-hydroxy-9, 11-octadecadienoic acid, 14, 15-epoxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, arachidonic acid and docosahexaenoic acid by 50 percent methanol aqueous solution, and mixing the components according to the component proportion of the following table 1 to prepare the standard substance mixed solution of which the final concentration of each component in the mixed solution is 1000 ng/ml;
table 1 arachidonic acid 15 standard mixed solutions were prepared with ingredient concentrations.
Step S2, the standard substance mixture obtained in step S1 is diluted with ethanol in a gradient concentration to a series of concentration standard substance mixtures of 10 concentration gradients, as shown in table 2 below.
TABLE 2 gradient dilution Table for arachidonic acid standard solution
Respectively adding 20 mu L of mixed internal standard working solution obtained in the step S1 and 500 mu L of methanol containing 1% of FA into each gradient concentration standard product mixed solution, carrying out 500W ultrasonic treatment in an ice bath for 30min, centrifuging for 10min at 14000g and 4 ℃, taking the supernatant, and carrying out liquid-liquid extraction, wherein the specific liquid-liquid extraction step is to use an Oasis HLB solid phase extraction 96-well plate, add 3mL of methanol activated well plates for 3 times, and add 1mL of methanol each time; adding 3mL of pure water balance pore plate for 3 times, wherein 1mL of pure water balance pore plate is added each time; adding the supernatant prepared in the step S2 or S3 into a pore plate; adding 2mL of cleaning solution A for 2 times, and adding 1mL of cleaning solution A for each time; adding 2mL of cleaning solution B for 2 times, and adding 1mL of cleaning solution B for each time; finally, adding 1mL of methanol to elute the sample to obtain eluted filtrate, and drying the eluted filtrate by using nitrogen for later use;
step S3, unfreezing the plasma sample at 4 ℃, taking 200 microliter of sample, adding 20 microliter of mixed internal standard working solution in the step S1 and 500 microliter of methanol containing 1% of FA, carrying out ultrasonic treatment for 30min at 500W in an ice bath, centrifuging for 10min at 4 ℃ by 14000g, taking supernatant, and carrying out liquid-liquid extraction, wherein the specific liquid-liquid extraction step is to use an Oasis HLB solid phase extraction 96 pore plate, add 3mL methanol activation pore plates for 3 times, and add 1mL each time; adding 3mL of pure water balance pore plate for 3 times, wherein 1mL of pure water balance pore plate is added each time; adding the supernatant prepared in the step S2 or S3 into a pore plate; adding 2mL of cleaning solution A for 2 times, and adding 1mL of cleaning solution A for each time; adding 2mL of cleaning solution B for 2 times, and adding 1mL of cleaning solution B for each time; finally, adding 1mL of methanol to elute the sample to obtain eluted filtrate, and drying the eluted filtrate by using nitrogen for later use;
step S4, samples obtained in step S2 and step S3 were mixed with 100ul acetonitrile: water: re-dissolving formic acid solution (80: 19: 1, v/v/v), performing high performance liquid chromatography-tandem mass spectrometry, loading sample amount of 4ul, performing chromatographic separation by adopting a Waters acquisition UPLC I-Class system, introducing sample at 4 ℃, column temperature of 35 ℃, flow rate of 400 muL/min, and sample amount of 2 muL; the liquid phase gradient is as follows, 0-1min, the B phase is maintained at 30%; 1-7min, the phase B changes linearly from 30% to 80%; 7-9min, phase B changes linearly from 80% to 90%; 9-11min, keeping phase B at 90%; 11-11.2min, phase B changes linearly from 90% to 30%; 11.2-14min, keeping phase B at 30%;
performing mass spectrometry by using 5500QTRAP mass Spectrometer (SCIEX) in negative ion mode, and using multiple reaction monitoring mode under the condition that the temperature of the ion source is 500 ℃; the atomization air pressure is 50; auxiliary air pressure: 50; air curtain air: 30, of a nitrogen-containing gas; spraying voltage: 4500V.
Each ion pair and its corresponding declustering voltage, collision voltage and collision cell exit voltage parameters are as in table 3 below.
Table 3 mass spectrometry ion pair declustering voltage, collision voltage and collision cell exit voltage parameter tables.
As a preferable example, the methanol containing 1% FA in steps S2 and S3 further contains an antioxidant solution of 2, 6-di-tert-butyl-p-cresol, and the preparation method is as follows, weighing 11.6mg of 2, 6-di-tert-butyl-p-cresol, adding 5mL of methanol to dissolve, adding 50. mu.L of formic acid, and mixing well.
As a preferred embodiment, the cleaning solution A is prepared by measuring 38mL of pure water, adding 2mL of 2, 6-di-tert-butyl-p-cresol and mixing; the cleaning solution B is prepared by measuring 20mL of the cleaning solution A and adding 200 mu L of formic acid for mixing.
The detection result of this embodiment is further analyzed to verify the feasibility of the method provided by the present invention:
and establishing a standard curve by adopting an isotope internal standard quantitative method and taking the concentration of the arachidonic acid standard as an x axis and the peak area ratio of the arachidonic acid standard to the internal standard as a y axis. The concentration of arachidonic acid in plasma was calculated from this curve and the results were as follows:
15 the arachidonic acid has good linear relation in respective concentration linear range, meets the quantitative requirement, and can rapidly, conveniently, stably and accurately quantitatively detect the arachidonic acid in the blood plasma.
The above embodiments are only used for illustrating but not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (6)
1. A method for detecting arachidonic acid and metabolites thereof in human plasma is characterized by comprising the following steps:
step S1, dissolving an isotope internal standard product arachidonic acid by using methanol, preparing a mixed internal standard working solution, dissolving the standard product arachidonic acid by using a methanol aqueous solution, and preparing a standard product mixed solution;
step S2, using ethanol to dilute the standard substance mixed liquor obtained in the step S1 into a series of concentration standard substance mixed liquor with a plurality of concentration gradients in a gradient concentration manner, wherein the concentration gradients are more than three; respectively adding the mixed internal standard working solution in the step S1 and methanol containing 1% of FA into the mixed solution of the standard substance with each gradient concentration, carrying out ice bath ultrasonic treatment, centrifuging at low temperature, and then taking the supernatant to carry out liquid-liquid extraction to obtain an eluted filtrate after extraction for later use;
step S3, taking a sample to be detected, adding mixed internal standard working solution and methanol containing 1% of FA into the sample to be detected by the same operation method of step S2, carrying out ice bath ultrasonic treatment, centrifuging at low temperature, taking supernatant, and carrying out liquid-liquid extraction to obtain extracted filtrate for later use;
step S4, carrying out high performance liquid chromatography-tandem mass spectrometry on the extracted samples obtained in the steps S2 and S3, wherein the high performance liquid chromatography conditions comprise sample introduction at 4 ℃, column temperature of 35 ℃, flow rate of 400 mu L/min and sample introduction amount of 2 mu L; the liquid phase gradient is as follows, 0-1min, the B phase is maintained at 30%; 1-7min, the phase B changes linearly from 30% to 80%; 7-9min, phase B changes linearly from 80% to 90%; 9-11min, keeping phase B at 90%; 11-11.2min, phase B changes linearly from 90% to 30%; 11.2-14min, keeping phase B at 30%;
the mass spectrum detection is in a multi-reaction monitoring mode, and the ion source condition is that the temperature of the ion source is 500 ℃; the atomization air pressure is 50; auxiliary air pressure: 50; air curtain air: 30, of a nitrogen-containing gas; spraying voltage: 4500V.
2. A method as claimed in claim 1, wherein the step S1 comprises mixing arachidonic acid as an isotope, including thromboxane B2-D4, leukotriene B4-D4, leukotriene D4-D5, prostaglandin F2 α -4, 8-isoprostane F2 α -D4, 6-keto-prostaglandin F1 α -D4, 12 hydroxyeicosatetraenoic acid-D8, arachidonic acid-D8, prostaglandin D2-D4, (9S,10E,12Z) -9-hydroxyoctadeca-10, 12-dienoic acid-D4, (9Z,11E) -13-hydroxy-9, 11-octadecadienoic acid-D4, 15 hydroxyeicosatetraenoic acid-D4 and docosahexenoic acid-D4, dissolving the respective components with methanol, diluting the respective components with water to a final concentration of the respective components in 200ng/ml, arachidonic acid-D4 and docosahexaenoic acid-D4, dissolving the respective concentrations of arachidonic acid, and its metabolites in water as a final concentration, including arachidonic acid B2-D, arachidonic acid, and docosahexaenoic acid-D4, and docosahexaenoic acid solutions as a final concentration, and docosahexaenoic acid solutions as solutions, as solutions of step S465, and as solutions of step S466-5, 6-5-6-5, 6-13-5-13, 13-5, 13-6-5.
3. The method of claim 1, wherein the methanol containing 1% FA in steps S2 and S3 further contains an antioxidant; the ice bath ultrasonic treatment is 500W ultrasonic treatment in an ice bath for 30 min; the low-temperature centrifugation is performed for 10min at 4 ℃ at 14000 g.
4. The method for detecting arachidonic acid and its metabolites in human plasma as claimed in claim 3, wherein the antioxidant is 2, 6-di-tert-butyl-p-cresol, which is prepared by weighing 2, 6-di-tert-butyl-p-cresol, dissolving in methanol at a ratio of 2.32mg/mL, adding 1/100 volumes of formic acid, and mixing well.
5. The method of claim 4, wherein the liquid-liquid extraction in steps S2 and S3 is performed by 3 times adding 1mL of 3mL methanol-activated well plates using Oasis HLB solid-phase extraction 96-well plates; adding 3mL of pure water balance pore plate for 3 times, wherein 1mL of pure water balance pore plate is added each time; adding the supernatant prepared in the step S2 or S3 into a pore plate; adding 2mL of cleaning solution A for 2 times, and adding 1mL of cleaning solution A for each time; adding 2mL of cleaning solution B for 2 times, and adding 1mL of cleaning solution B for each time; and finally, adding 1mL of methanol to elute the sample to obtain eluted filtrate.
6. The method for detecting arachidonic acid and its metabolites in human plasma according to claim 5, wherein the washing solution A is prepared by mixing the following raw materials in a volume ratio of 19: 1-water: the antioxidant is mixed in proportion; the cleaning solution B is prepared from the following components in percentage by volume of 10000: 1-cleaning liquid a: mixing with formic acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113049802A (en) * | 2021-03-23 | 2021-06-29 | 广州医科大学附属第一医院（广州呼吸中心） | Serological marker for evaluating allergen-specific immunotherapy effect |
| CN115876935A (en) * | 2022-12-29 | 2023-03-31 | 大连博源医学科技有限公司 | Method for detecting 11-dehydrothromboxane B in blood 2 Liquid chromatography-tandem mass spectrometry method and kit |
| CN115980223A (en) * | 2022-12-29 | 2023-04-18 | 大连博源医学科技有限公司 | Method for detecting 11-dehydrothromboxane B in blood 2 Liquid chromatography-tandem mass spectrometry method and kit |
| CN115876935B (en) * | 2022-12-29 | 2023-11-14 | 大连博源医学科技有限公司 | A method for detecting 11-dehydrothromboxane B in blood 2 Liquid chromatography-tandem mass spectrometry method and kit |
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