CN112858518A - Method for extracting acadesine from urine and detection method - Google Patents
Method for extracting acadesine from urine and detection method Download PDFInfo
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- 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
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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
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Abstract
The invention belongs to the technical field of extraction and analytical chemistry, and particularly relates to a method for extracting acarbosin in urine and a detection method, wherein the method for extracting acarbosin in urine comprises the following steps: (1) putting urine into a container, adding an acidifying reagent and enzyme, and performing enzymolysis at 50-60 deg.C for 1-3 h; (2) and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the enzyme-hydrolyzed enzyme-linked immunosorbent assay solution. The detection limit of the detection method is as low as 1ng/mL, the quantification limit is as low as 10ng/mL, and the detection method is not limited by different constitutions, ages, ethnicities and sports items in the detection process and has wide application range; the test method of the invention has the matrix effect of 89-104%, the extraction recovery rate of 87-106%, the standard deviation in the day of 1.4-13.8%, the standard deviation in the day of 1.3-16.3% and the average accuracy in three days of 98.3-100%. In conclusion, the detection method is simple to operate, has a wide linear range (1-10000ng/ml), and greatly meets the daily detection requirement.
Description
Technical Field
The invention belongs to the technical field of extraction and analytical chemistry, and particularly relates to a method for extracting acadesine from urine and a detection method.
Background
Acadesine (5-amino, 4-imidazole carboxamide nucleoside, AICAR), a class of important nitrogen-containing heterocyclic compounds, which are widely present in the body of mammals and in the synthesis of drug molecules, has the following structure:
the acadesine can be well used as a bioactive molecule to participate in chemical reactions due to the glycosyl fragment existing in the acadesine. One of the effects is that adenosine monophosphate activates protein kinase (AMPK) activator in the body of mammal, and reduces malonyl coenzyme a level in the body of mammal. In addition, the acadesine preparation can promote the organism to carry out continuous reaction, intervene metabolism by activating fatty acid oxidation, thereby increasing insulin sensitivity and having benefits on diseases such as diabetes, hypertension, atherosclerosis, polycystic ovary syndrome, gallstone and the like. Also, AICAR can be used in obesity treatment, which was found to promote lipogenesis with a positive effect on muscle formation by an exercise capacity increase test for three consecutive days, and chronic intermittent treatment of AICAR did not produce any significant toxic effects. Recent studies have shown that endurance performance is enhanced over time in untrained or untrained mice after administration of AICAR, known as "sports pills", and is therefore classified as a gene stimulant by the world anti-stimulant agency (WADA) and prohibited for use by athletes. Because of its ability to regulate exercise limitation and therapeutic efficacy of diseases, research on the synthesis, detection and further application of acadesine has received much attention. At present, laboratories in various countries use liquid chromatography-mass spectrometry to perform quantitative analysis on matrices such as urine, saliva and plasma. But since AICAR is endogenous, it is naturally present in the human body. The AICAR concentration level depends on different constitutions, ages, races, sports items, and the like, and uniform threshold establishment cannot be achieved at the time of method confirmation. The literature reports that the lowest detection concentration is 100ng/ml by a direct dilution method; after pretreatment optimization is carried out on the SPE column, the limit of quantification is 60ng/ml, and the normal requirement cannot be met, so that the detection method for the AICAR is in a continuous optimization development process.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a method for extracting acadesine from urine, which comprises the following steps:
(1) putting urine into a container, adding an acidifying reagent and enzyme, and performing enzymolysis at 50-60 deg.C for 1-3 h;
(2) and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the enzyme-hydrolyzed enzyme-linked immunosorbent assay solution.
In a preferred embodiment of the present invention, the acidifying reagent is a phosphate buffer.
In a preferred embodiment of the present invention, the phosphate buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 5.5 to 6.5.
In a preferred embodiment of the present invention, the precipitating agent is methanol and/or acetonitrile.
As a preferable technical scheme of the invention, the volume ratio of the urine to the acidifying reagent is (1-3): 1.
As a preferable technical scheme of the invention, the volume ratio of the enzymolysis liquid to the precipitator is (0.8-1.2): 1.
as a preferable technical scheme of the invention, the volume ratio of the urine to the enzyme is (18-22): 1.
the second aspect of the invention provides a method for detecting acadesine in urine, which comprises the following steps:
1) extracting acadesine from urine;
2) diluting the urine extract obtained in the step 1), and then carrying out liquid chromatography and mass spectrometry detection.
As a preferred embodiment of the present invention, the step 1) includes: adding urine into a container, adding an internal standard substance, an acidifying reagent and enzyme, and performing enzymolysis at 50-60 ℃ for 1-3 h; and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the urine extract.
As a preferable technical scheme of the invention, the chromatographic column for liquid chromatography detection is any one of reversed-phase C18, T3 and F5 chromatographic columns.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection limit of the detection method is as low as 1ng/mL, the quantification limit is as low as 10ng/mL, and the detection method is not limited by different constitutions, ages, ethnicities and sports items in the detection process and has wide application range;
(2) the detection method has the matrix effect of 89-104 percent, the extraction recovery rate of 87-106 percent, the standard deviation in the day of 1.4-13.8 percent, the standard deviation in the day of 1.3-16.3 percent and the average accuracy in three days of 98.3-100 percent;
(3) the glucuronidase can completely hydrolyze the glucocorticoid combined with glucose, so that the glucocorticoid exists in a free state;
(4) in the application, when the phosphate buffer solution is controlled to be 6-6.9, particularly 6.84, the enzymolysis is complete during later enzymolysis, and the quantitative detection of acadesine is more accurate;
(5) in the application, methanol is used as a precipitator, and the reproducibility is good when the urine extract is diluted and detected in the later period.
Drawings
FIG. 1 is a standard curve obtained by plotting the graph of example 1 of the present invention;
FIG. 2 is a detection spectrum of the detection limit concentration in example 1 of the present invention;
FIG. 3 is a detection spectrum of quantitative limiting concentration in example 1 of the present invention;
FIG. 4 is a standard curve chart obtained by plotting in example 4 of the present invention.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The invention provides a method for extracting acadesine from urine, which comprises the following steps:
(1) putting urine into a container, adding an acidifying reagent and enzyme, and performing enzymolysis at 50-60 deg.C for 1-3 h;
(2) and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the enzyme-hydrolyzed enzyme-linked immunosorbent assay solution.
In one embodiment, the acidifying reagent is a phosphate buffer.
Preferably, the phosphate buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 6-6.9; more preferably, the phosphate buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 6.84.
In the application, when the phosphate buffer solution is controlled to be 6-6.9, particularly 6.84, the enzymolysis is complete in later enzymolysis, and the quantitative detection of acadesine is more accurate.
In one embodiment, the precipitating agent is methanol and/or acetonitrile.
Preferably, the precipitant is methanol.
In the application, methanol is used as a precipitator, and the reproducibility is good when the urine extract is diluted and detected in the later period.
In one embodiment, the volume ratio of urine to acidifying agent is (1-3): 1.
preferably, the volume ratio of the urine to the acidifying agent is 2: 1.
in one embodiment, the volume ratio of the enzymatic hydrolysate to the precipitant is (0.8-1.2): 1.
preferably, the volume ratio of the enzymolysis liquid to the precipitating agent is 1: 1.
in one embodiment, the volume ratio of urine to enzyme is (18-22): 1.
preferably, the volume ratio of urine to enzyme is 20: 1.
the enzyme of the present invention is not particularly limited and may be routinely selected by those skilled in the art.
In one embodiment, the enzyme is glucuronidase.
The glucuronidase enzyme described herein allows complete hydrolysis of the glucose-bound glucocorticoid, leaving it in its entirety in the free form.
In one embodiment, the step (1) comprises: putting urine into a container, adding an acidifying reagent and enzyme, mixing uniformly, and carrying out enzymolysis for 2h in water bath at 55 ℃.
In one embodiment, the step (2) comprises: after the enzymolysis is finished, obtaining enzymolysis liquid, cooling to room temperature, shaking at the rotating speed of 2000-.
In a preferred embodiment, the step (2) comprises: and after the enzymolysis is finished, obtaining enzymolysis liquid, cooling to room temperature, shaking at the rotating speed of 2500rpm for 10s, adding a precipitator, centrifuging at the rotating speed of 13000rpm for 3min at 4 ℃, and taking supernatant to obtain the enzyme-linked immunosorbent assay.
The second aspect of the invention provides a method for detecting acadesine in urine, which comprises the following steps:
1) extracting acadesine from urine;
2) diluting the urine extract obtained in the step 1), and then carrying out liquid chromatography and mass spectrometry detection.
In one embodiment, the step 1) comprises: adding urine into a container, adding an internal standard substance, an acidifying reagent and enzyme, and performing enzymolysis at 50-60 ℃ for 1-3 h; and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the urine extract.
Preferably, the volume ratio of the urine to the internal standard substance is (18-22): 1; more preferably, the volume ratio of the urine to the internal standard is 20: 1.
in a preferred embodiment, the step 1) includes: adding urine into a container, adding an internal standard substance, an acidifying reagent and enzyme, and performing enzymolysis at 50-60 ℃ for 1-3 h; after the enzymolysis is finished, obtaining enzymolysis liquid, cooling to room temperature, shaking at the rotating speed of 2000-.
In a more preferred embodiment, the step 1) comprises: adding urine into a container, adding an internal standard substance, an acidifying reagent and enzyme, and carrying out enzymolysis at 55 ℃ for 2 h; and after the enzymolysis is finished, obtaining enzymolysis liquid, cooling to room temperature, shaking at the rotating speed of 2500rpm for 10s, adding a precipitator, centrifuging at the rotating speed of 13000rpm for 3min at 4 ℃, and taking supernatant to obtain the urine extract.
In one embodiment, in the step 2), the diluting solvent of the urine extract is water.
Preferably, the volume ratio of the urine extract to water is 1: (3-5); more preferably, the volume ratio of the urine extract to water is 1: 4.
in one embodiment, the column used for purification is selected from any one of reverse C18, T3, F5 columns.
Preferably, the chromatographic column is an F5 chromatographic column.
In one embodiment, the mobile phase is water when the chromatography column is purifying.
Preferably, the mobile phase comprises water and formic acid; further preferably, the volume ratio of formic acid to water is 1: (990-1100); more preferably, the volume ratio of formic acid to water is 1: 1000.
in one embodiment, the flow rate of the mobile phase is from 0.1 to 0.5 mL/min.
Preferably, the flow rate of the mobile phase is 0.3 mL/min.
In one embodiment, the column temperature of the chromatography column is 10-45 ℃.
Preferably, the column temperature of the chromatographic column is 40 ℃.
In one embodiment, the column purification is 100% isocratic elution with an elution time of 2-8 min.
Preferably, the elution time is 4 min.
In one embodiment, the mass spectrometry conditions are: electrospray ion source (ESI); a positive ion detection mode; capillary voltage: 4000V; ion source temperature: 350 ℃; flow rate of the dryer: 10L/min.
AICAR-internal standard structure as follows:
examples
Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that these examples are merely illustrative and not restrictive. The starting materials used in the examples which follow are all commercially available unless otherwise stated.
Example 1
The embodiment 1 of the invention provides a method for detecting acadesine in urine, which comprises the following specific steps:
(1) extracting acadesine from urine:
putting 200uL urine into a 2mLPE tube, adding 10uL internal standard, 100uL Phosphate Buffer Solution (PBS) and 10uL enzyme, uniformly mixing, and carrying out enzymolysis for 2h at 55 ℃ in a water bath; cooling to room temperature after enzymolysis, shaking (at 2500rpm for 10 s), collecting 200uL mixed solution, adding 200uL methanol, centrifuging for 3min at 4 deg.C and 13000 rpm; after centrifugation, taking 200uL of supernatant urine extract, adding 800uL of water, uniformly mixing to obtain urine sample detection liquid, and detecting; the phosphate buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value is 6.84; the enzyme is glucuronidase.
(2) Liquid chromatography detection:
performing liquid chromatography detection analysis on the urine sample detection solution obtained in the step (1), wherein the conditions are as follows: a chromatographic column: f5 chromatography columns (waters); mobile phase: water (containing 1 vol% formic acid); flow rate: 0.3 mL/min; sample introduction amount: 2 uL; column temperature: at 40 ℃.
(3) Mass spectrum detection:
carrying out mass spectrum detection analysis on the urine sample detection solution obtained in the step (1), wherein the conditions are as follows: electrospray ion source (ESI); a positive ion detection mode; capillary voltage: 4000V; ion source temperature: 350 ℃; flow rate of the dryer: 10L/min.
| Compound (I) | Parent ion | Daughter ions | Collision voltage |
| AICAR | 259.1 | 127.1 | 15V |
| AICAR | 259.1 | 110.1 | 27V |
| AICAR | 259.1 | 242.2 | 15V |
| AICAR-internal standard | 262.1 | 130.2 | 16V |
| AICAR-internal standard | 262.1 | 113.0 | 24V |
| AICAR-internal standard | 262.1 | 245.1 | 15V |
AICAR-internal standard structure as follows:
whether the sample is the same substance is judged by comparing peak retention time in the blank urine sample plus the acadesine internal standard with peak retention time in the acadesine internal standard in pure water and relative abundance ratio of each ion pair, and the result shows that other substances in urine can not interfere the detection of the acadesine internal standard, which indicates that the detection method provided by the invention can meet the selectivity requirement in daily detection.
Preparation of a standard curve:
1ng/mL, 10ng/mL, 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL, 10000ng/mL are prepared respectively. And performing linear regression on the obtained data by taking the concentration X (ng/mL) of the standard substance as a horizontal coordinate and taking the peak area ratio Y of the corresponding peak area to the internal standard peak area as a vertical coordinate to obtain a regression equation and a correlation coefficient. The mother liquor is 1mg/mL alcafurthin methanol solution, and the solvent used in the dilution process is water. 2 batches of linear standard curves were made in parallel,FIG. 1 is the resulting standard curve plot, R20.99831, meeting daily detection requirements.
Detection limit and quantification limit testing: the lowest detection Limit (LOD) was confirmed by the signal-to-noise ratio method (S/N > 3); the limit of quantitation (LOQ) requires that precision and accuracy be achieved while meeting the minimum detection concentration of the signal-to-noise ratio method (S/N > 10). FIG. 2 is a detection spectrum of detection limit concentration, FIG. 3 is a detection spectrum of quantitative limit concentration, and the detection limit of the present invention is 1ng/mL and the quantitative limit is 10ng/mL obtained by analysis from FIGS. 2 and 3.
And (3) testing precision and accuracy: preparing a dilute solution of acadesine: urine samples with high (5000ng/mL), medium (1000ng/mL), low (100ng/mL) and ultra-low (10ng/mL) concentrations were selected, and 4 samples were subjected to the pretreatment method described in this example and subjected to assay analysis, with the Relative Standard Deviation (RSD) of each concentration sample per day as the in-day precision. Three concentrations of high, medium and low are prepared continuously for 2 days, 10 repeated quality control samples are detected at each concentration point, and the RSD of each concentration sample is calculated to be used as the daytime precision. The percent of the average concentration value of the quality control sample and the added concentration is taken as the accuracy. The precision and accuracy criteria are that RSD is within + -20% around LOQ and within + -15% above LOQ. The intra-day precision and the inter-day precision were calculated. The standard deviation in the day was calculated to be 1.4-13.8% (RSD < 15%); standard deviation between days 1.3-16.3% (RSD < 20%). The average accuracy of three days is 98.3-100%, which meets the requirement of the verification method.
Recovery and matrix effect testing: preparing a dilute solution of acadesine: urine samples with high (5000ng/mL), medium (1000ng/mL), low (100ng/mL) and ultra-low (10ng/mL) concentrations were selected, and 4 samples were subjected to the pretreatment method described in this example and subjected to detection analysis.
Group 1: taking 4 groups of blank urine samples, adding 4 samples in each group, respectively adding standard samples with high (5000ng/mL), medium (1000ng/mL), low (100ng/mL) and ultralow (10ng/mL) concentrations and a certain amount of internal standard, processing and detecting according to a pretreatment method, and measuring a response value A.
Group 2: 4 groups of blank urine samples are taken, 4 samples in each group are treated according to the pretreatment method in the embodiment, after enzymolysis, standard samples with high (5000ng/mL), medium (1000ng/mL), low (100ng/mL) and ultra-low (10ng/mL) concentrations and a certain amount of internal standard are respectively added, and then drying, redissolution, detection and analysis are carried out. A response value B was measured.
Group 3: directly using water to prepare the solution with the same concentration as the above four solutions, directly injecting the sample, and measuring the response value C.
Calculated according to the following formula: the extraction recovery rate is A/B × 100%, and the matrix effect is B/C × 10%.
The matrix effect obtained by final detection is 89-104%; the extraction recovery rate is 87-106%, and the requirement of the verification method is met.
Example 2
The embodiment 2 of the invention provides a method for detecting acadesine in urine, which is implemented in the same way as the embodiment 1, except that the pH value of the phosphate buffer solution is 7.2.
The standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.84078, LOD 5ng/mL, LOQ 20 ng/mL.
Example 3
The embodiment 3 of the invention provides a method for detecting acadesine in urine, which is implemented in the same way as the embodiment 1, except that the pH value of the phosphate buffer solution is 5.5.
The standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.80412, LOD 8ng/mL, LOQ 30 ng/mL.
Example 4
The embodiment 4 of the invention provides a method for detecting acadesine in urine, which is implemented in the same way as the embodiment 1, except that the chromatographic column is a T3 chromatographic column.
The standard curve drawing, detection limit and quantitative limit testing method is the same as that of example 1, the standard curve is shown in figure 4, and R is obtained2Is 0.99558. LOD was 2ng/mL and LOQ was 12 ng/mL.
Example 5
The embodiment 5 of the invention provides a method for detecting acadesine in urine, which is implemented in the same way as the embodiment 1, except that after centrifugation is finished, 200uL of supernatant urine extract is taken, and 1000uL of water is added.
The standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained2Is 0.99713. LOD is 1ng/mL, LOQ is 15 ng/mL.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (10)
1. A method for extracting acadesine from urine is characterized by comprising the following steps:
(1) putting urine into a container, adding an acidifying reagent and enzyme, and performing enzymolysis at 50-60 deg.C for 1-3 h;
(2) and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the enzyme-hydrolyzed enzyme-linked immunosorbent assay solution.
2. The method for extracting acadesine from urine according to claim 1, wherein the acidifying reagent is phosphate buffer.
3. The method for extracting acadesine from urine as claimed in claim 2, wherein the phosphate buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 6-6.9.
4. The method for extracting acadesine from urine according to any of claims 1-3, wherein the precipitating agent is methanol and/or acetonitrile.
5. The method for extracting acadesine from urine according to any of claims 1 to 3, wherein the volume ratio of the urine to the acidifying agent is (1-3): 1.
6. the method for extracting acadesine from urine as claimed in claim 5, wherein the volume ratio of the enzymolysis solution to the precipitant is (0.8-1.2): 1.
7. the method for extracting acadesine from urine as claimed in claim 5, wherein the volume ratio of the urine to the enzyme is (18-22): 1.
8. a method for detecting acadesine in urine is characterized by comprising the following steps:
1) extracting acadesine from urine by the method of any one of claims 1 to 7;
2) diluting the urine extract obtained in the step 1), and then carrying out liquid chromatography and mass spectrometry detection.
9. The method for detecting acadesine in urine according to claim 8, wherein the step 1) comprises: adding urine into a container, adding an internal standard substance, an acidifying reagent and enzyme, and performing enzymolysis at 50-60 ℃ for 1-3 h; and after enzymolysis is finished, obtaining enzymolysis liquid, cooling, shaking, adding a precipitator, centrifuging, and taking supernate to obtain the urine extract.
10. The method for detecting acadesine in urine according to claim 9, wherein the chromatographic column for liquid chromatography detection is any one of reversed-phase C18, T3 and F5 chromatographic columns.
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| CN115267000A (en) * | 2022-08-05 | 2022-11-01 | 吴昕辰 | Method for detecting acadesine stimulant |
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