CN112946109A - Detection method and application of endogenous glucocorticoid - Google Patents
Detection method and application of endogenous glucocorticoid 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
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a detection method of endogenous glucocorticoid and application thereof, wherein the detection method of the endogenous glucocorticoid comprises the following steps: the method comprises the steps of optimizing extraction conditions of several endogenous glucocorticoids in a urine sample, establishing a mass spectrum detection process and analyzing the population value of a sports training sample. The LOD obtained by the method for detecting endogenous glucocorticoid provided by the invention is 0.1ng/mL, the LOQ is 1ng/mL, the extraction recovery rate is more than 87%, and the matrix effect, the precision and the accuracy are all within +/-15%. The method provided by the invention can meet the daily detection requirement, and meanwhile, the high-flux sample pretreatment can be realized during the actual sample detection, so that the time cost is saved, the manual operation error is reduced, and the reproducibility and the stability are improved.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a detection method of endogenous glucocorticoid and application thereof.
Background
Glucocorticoid (GC) is an extremely important regulatory molecule in the body, plays an important role in regulating development, growth, metabolism, immune function and the like of the body, is the most important regulatory hormone for stress response of the body, and is also the most widely and effectively anti-inflammatory and immunosuppressant in clinic. Glucocorticoids are often preferred in emergency or critical situations. The clinically common glucocorticoid medicaments comprise prednisone, methylprednisolone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone and the like, have various effects of resisting toxicity, allergy, shock, nonspecific inhibition of immunity, defervescence and the like, can prevent and stop immune inflammatory reaction and pathological immune reaction, and are almost effective on any type of allergic diseases. Several common glucocorticoid drugs have the following structure:
in addition, clinical studies show that glucocorticoids have anti-inflammatory and injury repairing effects in different degrees, can enhance exercise endurance and explosive force and improve exercise performance, so that the glucocorticoids are listed as stimulants by the world wide anti-excitant agency (WADA) and are prohibited for athletes. At present, laboratories in various countries use detection means such as LC-MS, GC-MS, liquid chromatography, gas mass spectrometry and the like to perform qualitative and quantitative analysis on matrixes such as urine, saliva, plasma and the like. However, since cortisone and hydrocortisone are endogenous and naturally exist in the human body, and the concentration level thereof depends on different constitutions, ages, races, sports items, and the like, uniform threshold establishment cannot be achieved in the process of method confirmation. Based on this, chemists typically represent prednisone and prednisolone from the perspective of their metabolites, and since the concentrations of the metabolites are more dependent on the maternal concentration and do not fluctuate too much in a short time, the monitoring of these two substances can ensure the fairness and justice of sports competition. The official documents of the international antiperspirants agency at present require that the concentration of prednisone and prednisolone is not more than 60ng/ml, if the detection concentration is between 30 and 60ng/ml, the source of the prednisone and prednisolone needs to be further clarified by using isotope ratio mass spectrometry, if the endogenous source is determined to be generated by the antiperspirants agency, if the determination result shows that the external introduction is performed, the antiperspirants are determined to be violated, and certain abstaining penalty is generated. Because of its ability to regulate exercise limitation and disease treatment efficacy, research on synthesis, detection and further application of glucocorticoids has received much attention. However, the existing glucocorticoid detection has poor sensitivity and high quantitative limit, and cannot be applied to the detection of people of different ethnicities, different ages and different constitutions.
Disclosure of Invention
In view of the problems in the prior art, the first aspect of the present invention provides a method for detecting endogenous glucocorticoid, comprising the following steps:
(1) extraction of glucocorticoids: putting the biological liquid into a container, adding an internal standard substance, an acidifying reagent and enzyme, and carrying out enzymolysis for 1-3h at 50-60 ℃; adding alkali liquor and ether substances after enzymolysis, shaking, centrifuging, collecting supernatant, and adding N2Drying, adding the extracting solution for redissolving to obtain a solution to be detected;
(2) and (4) carrying out liquid chromatography and mass spectrum detection on the liquid to be detected, and analyzing to obtain the product.
As a preferable technical scheme of the invention, the alkali liquor is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate.
As a preferable technical scheme of the invention, the alkali liquor is sodium bicarbonate and sodium carbonate, and the pH of the alkali liquor is 9-10.
In a preferred embodiment of the present invention, the ether-based substance has 4 to 8 carbon atoms.
As a preferable technical scheme of the invention, the volume ratio of the ether substance to the alkali liquor is (70-90): 1.
as a preferred technical scheme of the invention, the volume ratio of the biological fluid to the internal standard substance is (8-12): 1.
in a preferred embodiment of the present invention, the extraction solution is water and acetonitrile.
As a preferable technical scheme of the invention, the volume ratio of the water to the acetonitrile is (2-6): 1.
in a preferred embodiment of the present invention, in the liquid chromatography detection, the liquid chromatography column is selected from any one of reversed-phase C18, T3 and F5 columns.
The invention provides an application of the method for detecting endogenous glucocorticoid in quantitative detection of glucocorticoid in urine, saliva and plasma.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection method of endogenous glucocorticoid provided by the invention has good selectivity, other substances in the matrix can not interfere with the determination of endogenous glucocorticoid (cortisone, hydrocortisone, prednisone and prednisolone), and the LOD and LOQ of four mixed glucocorticoids (hydrocortisone, cortisone, prednisone and prednisolone) are respectively 0.1ng/mL and 1 ng/mL. The matrix effect is 84.35-97.68%; the extraction recovery rate is more than 87%; the precision in the day is less than 7.8%; the daytime precision is less than 5.71%; the accuracy is 93.59-107.71%, the verification result of the methodology is enough to meet daily detection requirements, meanwhile, automatic processing can be realized during actual sample detection and analysis, the automation degree of the whole process can be increased, assembly line operation can be realized, the time cost is saved, manual operation errors are reduced, and the repeatability and the stability are improved.
(2) In this application, control alkali lye for sodium bicarbonate and sodium carbonate, and pH is 9.5, makes things convenient for later stage centrifugation effect, and it is easy to get the supernatant after the centrifugation finishes.
(3) By controlling the ether substance to be methyl tert-butyl ether, the volume ratio of the ether substance to the alkali liquor is (70-90): 1, the obtained liquid to be detected is not limited by people of different ethnicities, different ages and different constitutions during subsequent liquid chromatography and mass spectrometry detection and analysis.
Drawings
FIG. 1 is a standard curve of hydrocortisone plotted in example 1 of the present invention;
FIG. 2 is a quantitative and qualitative selective ion chromatogram of hydrocortisone detected in example 1 of the present invention;
FIG. 3 is a standard curve of cortisone obtained by the method of example 1;
FIG. 4 is a quantitative and qualitative selective ion chromatogram of cortisone detected in example 1 of the present invention;
FIG. 5 is a standard curve diagram of prednisone obtained by the method of example 1;
FIG. 6 is a quantitative and qualitative selective ion chromatogram of prednisone detected in example 1 of the present invention;
FIG. 7 is a standard curve diagram of prednisolone obtained by the method of example 1;
FIG. 8 is a quantitative and qualitative selective ion chromatogram of prednisolone obtained by detection in example 1 of the present invention;
FIG. 9 is a spectrum of detection of the detection limit concentration of hydrocortisone in example 1 according to the present invention;
FIG. 10 is a spectrum of the limit concentration of hydrocortisone in the example 1;
FIG. 11 is a diagram of an automated inspection apparatus of the present invention;
FIG. 12 is a graph showing the change in cortisone concentration before and after exercise;
FIG. 13 is a graph showing the change in cortisol concentration before and after exercise;
FIG. 14 is a graph of prednisone concentration change before and after exercise;
fig. 15 is a graph of prednisolone concentration changes before and after exercise.
Detailed Description
The invention realizes the innovation of a detection technology, and the specific implementation process depends on the following embodiments of four specific glucocorticoids for detailed description. The procedures, conditions, experiments, methodological verifications, and the like, which are mentioned in the practice of the present invention, are general knowledge and common general knowledge in the art, except for the information specifically mentioned below, and the present invention is not particularly limited.
In a first aspect, the present invention provides a method for detecting endogenous glucocorticoid, comprising the following steps:
(1) extraction of glucocorticoids: putting the biological liquid into a container, adding an internal standard substance, an acidifying reagent and enzyme, and carrying out enzymolysis for 1-3h at 50-60 ℃; adding alkali liquor and ether substances after enzymolysis, shaking, centrifuging, collecting supernatant, and adding N2Drying, adding the extracting solution for redissolving to obtain a solution to be detected;
(2) and (4) carrying out liquid chromatography and mass spectrum detection on the liquid to be detected, and analyzing to obtain the product.
In one embodiment, the alkali solution is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate.
Preferably, the alkali liquor is sodium bicarbonate and sodium carbonate, and the pH value of the alkali liquor is 9-10; more preferably, the pH of the lye is 9.5.
In this application, control alkali lye for sodium bicarbonate and sodium carbonate, and pH is 9.5, makes things convenient for later stage centrifugation effect, and it is easy to get the supernatant after the centrifugation finishes.
In one embodiment, the ether species has from 4 to 8 carbon atoms.
Preferably, the ether substance has 5 carbon atoms; more preferably, the ether substance is methyl tert-butyl ether.
In one embodiment, the volume ratio of the ether substance to the alkali liquor is (70-90): 1.
preferably, the volume ratio of the ether substances to the alkali liquor is 80: 1.
the ether substance is controlled to be methyl tert-butyl ether, and the volume ratio of the ether substance to the alkali liquor is (70-90): 1, the subsequent liquid chromatography and mass spectrometry detection and analysis of the obtained liquid to be detected are not limited by people of different ethnicities, different ages and different constitutions.
In one embodiment, the volume ratio of the biological fluid to the internal standard is (8-12): 1.
preferably, the volume ratio of the biological fluid to the internal standard substance is 10: 1.
in one embodiment, the extraction solution is water and acetonitrile.
Preferably, the volume ratio of the water to the acetonitrile is (2-6): 1; more preferably, the volume ratio of water to acetonitrile is 4: 1.
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 6.75.
In one embodiment, the volume ratio of the biological fluid to the acidifying agent is (1-3): 1.
preferably, the volume ratio of the biological fluid to the acidifying agent is 2: 1.
in one embodiment, the volume ratio of the biological fluid to the enzyme is (18-22): 1.
preferably, the volume ratio of the biological fluid to the enzyme is 20: 1.
in one embodiment, the volume ratio of the supernatant to the extract is (10-15): 1.
preferably, the volume ratio of the supernatant to the extracting solution is 12: 1.
in one embodiment, the step (1) comprises: putting the biological liquid into a container, adding an internal standard substance, an acidifying reagent and an enzyme, uniformly mixing, and carrying out enzymolysis for 1-3h at 50-60 ℃; adding alkali solution and ether substances after enzymolysis, shaking at 3000rpm 2000-5 min, centrifuging at 10000-15000rpm 1-5 deg.C for 10-20min, collecting supernatant, and centrifuging with N2Drying, adding the extracting solution for redissolving to obtain the solution to be detected.
In a preferred embodiment, the step (1) comprises: putting the biological fluid into a container, adding an internal standard substance, an acidifying reagent and an enzyme, uniformly mixing, and carrying out enzymolysis for 2 hours at 55 ℃; adding alkali solution and ether substances, shaking at 2500rpm for 3min, centrifuging at 4 deg.C and 13000rpm for 15min, collecting supernatant, and centrifuging to obtain N2Drying, adding the extracting solution for redissolving to obtain the solution to be detected.
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.
The internal standard substance is an internal standard substance of glucocorticoid.
In one embodiment, the liquid chromatography assay is one wherein the liquid chromatography column is selected from any of reverse-phase C18, T3, F5.
Preferably, the liquid chromatography column is a T3 chromatography column.
The chromatographic columns described in the present application are all available from waters corporation.
In one embodiment, the mobile phase a is water and formic acid in a liquid chromatography assay.
Preferably, the volume ratio of the water to the formic acid is (900- & lt 1100- & gt): 1; more preferably, the volume ratio of water to formic acid is 1000: 1.
in one embodiment, the mobile phase a further comprises ammonium acetate in the liquid chromatography assay.
Preferably, the concentration of the ammonium acetate in the mobile phase A is 0.8-1.2M; more preferably, the concentration of the ammonium acetate in the mobile phase a is 1M.
In one embodiment, the mobile phase B is acetonitrile in a liquid chromatography assay.
By controlling the specific mobile phase A and the specific mobile phase B, the method can obtain lower detection limit and quantitative phase, and expands the applicability of the detection method.
In one embodiment, the liquid chromatography assay is performed with a gradient elution gradient for a time period of 5-10 min.
Preferably, the elution time is 8 min.
In one embodiment, the mobile phase flow rate is from 0.1 to 0.5mL/min in a liquid chromatography assay.
Preferably, the mobile phase flow rate is 0.3 mL/min.
In one embodiment, the column temperature is from 10 ℃ to 45 ℃ in the liquid chromatography assay.
Preferably, the column temperature is 40 ℃.
In one embodiment, in the liquid chromatography assay, a sample volume of 5 μ l is taken and the analysis time is 8 min. The autosampler was maintained at 10 ℃.
In one embodiment, the gradient elution conditions are: 0-1 min, 20 vol% B; 20 → 70 vol% B for 1-4 min; 4-5.5 min, 70 → 95 vol% B; 5.5-6.2 min, 95 vol% B; 6.2-6.3 min, 95% → 20 vol% B; 6.3-8 min, 20 vol% B.
In one embodiment, the mass spectrometric detection conditions are: electrospray ion sources employ positive ion mode (ESI +); capillary voltage: 4000V; the ion source temperature is 350 ℃; the flow rate of the drying gas is 6L/min; atomizing: 45 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM) mode.
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 endogenous glucocorticoid, which comprises the following specific steps:
(1) extraction of glucocorticoids:
adding 20 mu L of internal standard substance, 100 mu L of Phosphate Buffered Saline (PBS) and 10 mu L of enzyme into 200uL of urine, uniformly mixing, and carrying out enzymolysis for 2h at 55 ℃ in a water bath; after the enzymolysis is finished, adding 10 mu L of alkali liquor and 800 mu L of methyl tert-butyl ether, and oscillating (rotating speed of 2500 rpm) for 3 min; centrifuging for 15min at 4 deg.C and 13000rpm, collecting supernatant 600 μ L, N2Drying, adding 50 mu L of extracting solution (V water: V acetonitrile 4:1) for redissolving to obtain a urine detection sample, and detecting; the phosphate buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value is 6.75; the enzyme is glucuronidase; the alkali liquor is sodium bicarbonate and potassium carbonate, and the pH is 9.5.
(2) Liquid chromatography detection:
performing liquid chromatography detection on the urine detection sample obtained in the step (1), wherein the conditions are as follows:
the liquid chromatography column is a Waters T3 column (2.1X 100mm,1.8 μm); the column temperature is 40 ℃; mobile phase a was composed of (water, 1M ammonium acetate and 0.1 vol% formic acid) and phase B (acetonitrile). Gradient elution; the flow rate is 0.3 mL/min; column temperature: 40 ℃; the sample volume is 5 μ L, and the analysis time is 8 min. The autosampler was maintained at 10 ℃. The gradient elution conditions were: 0-1 min, 20% B; 1-4 min, 20 → 70% B; 4-5.5 min, 70 → 95% B; 5.5-6.2 min, 95% B; 6.2-6.3 min, 95% → 20% B; 6.3-8 min, 20% B.
(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 sources employ positive ion mode (ESI +); capillary voltage: 4000V; the ion source temperature is 350 ℃; the flow rate of the drying gas is 6L/min; atomizing: 45 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM) mode.
FIG. 2 is a chromatogram of the quantitative and qualitative selection of hydrocortisone obtained by detection; FIG. 4 is a quantitative and qualitative selection ion chromatogram of cortisone obtained by detection; FIG. 6 is a quantitative and qualitative selective ion chromatogram of prednisone obtained by detection; fig. 8 is a quantitative and qualitative selective ion chromatogram of prednisolone obtained by detection.
Whether the sample is the same substance is judged by comparing peak retention time in a blank urine sample with a glucocorticoid internal standard and a glucocorticoid internal standard in pure water with relative abundance ratios of ion pairs, and the result shows that other substances in urine do not interfere with the detection of the glucocorticoid internal standard, so that the detection method provided by the invention can meet the selectivity requirement in daily detection.
Drawing a standard curve:
1ng/mL, 2ng/mL, 5ng/mL,10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 250ng/mL, 300ng/mL were prepared, respectively. The mother liquor is 1mg/ml glucocorticoid methanol solution, and the solvent used for preparing the solution in the gradient dilution process is methanol. And performing LC-MS (liquid chromatography-mass spectrometry) determination on each concentration sample, taking the concentration X (ng/mL) as a horizontal coordinate and taking the peak area ratio Y of the target object to the peak area of the internal standard as a vertical coordinate, performing linear regression, and automatically processing to obtain a regression equation and a correlation coefficient. R2Are all larger than 0.995, and meet the daily detection requirement. The hydrocortisone and standard curve maps are respectively shown in figures 1, 3, 5 and 7, and according to figures 1, 3, 5 and 7, the fitting result data are as follows:
detection limit and quantification limit testing:
the lowest detection limit is confirmed by a signal-to-noise ratio method (S/N is more than or equal to 3); the quantitation limit needs to be done with precision and accuracy while meeting the minimum detected concentration of the signal-to-noise ratio method (S/N > 10). Fig. 9 is a detection spectrum of the detection limit concentration of hydrocortisone, and fig. 10 is a detection spectrum of the quantitative limit concentration of hydrocortisone.
The LOD obtained by the method is 0.1ng/mL, and the LOQ is 1 ng/mL. Compared with the minimum quantitative limit of 60ng/mL in the researched literature, the detection method provided by the invention has ultrahigh sensitivity and can completely meet the daily detection requirement.
And (3) testing precision and accuracy: glucocorticoid diluted solutions were prepared according to the method of the Standard curve in this example, and the solutions were prepared to have high (250ng/mL), medium (20ng/mL) and low (1ng/mL), 10 parts of them were pretreated in this example in parallel and analyzed, and the Relative Standard Deviation (RSD) of each concentration sample per day was used as the precision in the day. 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 precision and accuracy are shown in the following table:
recovery and matrix effect testing: glucocorticoid diluted solutions were prepared according to the method of the standard curve preparation process in this example, urine samples with high (250ng/mL), medium (20ng/mL) and low (1ng/mL) concentrations were prepared, and 3 samples were pretreated and analyzed.
Group 1: 3 blank urine samples were taken, 6 samples per group, high (250ng/mL) and medium (20ng/mL) concentration standards and a certain amount of internal standard were added, and the samples were processed and analyzed according to the pretreatment method of this example to determine the response A.
Group 2: taking 3 groups of blank urine samples, 6 in each group, treating according to a pretreatment method, respectively adding high (250ng/mL) and medium (20ng/mL) concentration standard samples and a certain amount of internal standard after enzymolysis, and then drying, redissolving, detecting and analyzing. 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%.
Finally detecting the matrix effect obtained by the detection; the extraction recovery rate is more than 87%, and the requirement of the verification method is met.
The treatment was performed as a pretreatment method for evaluation of extraction recovery and matrix effect. Directly preparing the standard solution with the concentration by using water, and regarding the peak area as A; adding high (250ng/mL) and medium (20ng/mL) concentration standard samples into blank urine matrixes from six different sources respectively, and treating, detecting and analyzing according to a pretreatment method to obtain a response value B; the test was performed using blank urine from six different persons and adding standards of the same concentration, and the analysis results were as follows.
Automatically detecting an actual urine sample: the automatic control system is combined with the current artificial intelligence trend, the automation degree of the whole process setting is higher, the assembly line operation can be realized, the time cost is saved, the manual operation error is reduced, and the repeatability and the stability are improved. The detection of two actual samples before and after exercise is completed by collecting the personnel who perform exercise training, the graph 12 is a cortisone concentration change graph before and after exercise, the graph 13 is a cortisol concentration change graph before and after exercise, the graph 14 is a prednisone concentration change graph before and after exercise, the graph 15 is a prednisolone concentration change graph before and after exercise, and the analysis result shows that the in vivo content of cortisone is 2.1-124.6ng/mL, and the average value is 53.69 ng/mL; the in vivo content of hydrocortisone is 0.37-72.37ng/mL, and the average value is 20.92 ng/mL; the in vivo content range of prednisone is 0.16-8.88ng/mL, and the average value is 2.71 ng/mL; the in vivo content range of prednisolone is 0-1.64ng/mL, and the average value is 0.37 ng/mL. Prednisone and prednisolone, which are further metabolites, are relatively low in concentration in vivo, and therefore, the determination of the content of both substances in the international agency for anti-excitant above 60ng/ml is positive (indicating that athletes violate the anti-excitant rules and use the related prohibited drugs to achieve the effect of improving exercise).
Fig. 11 is a diagram of an automated inspection apparatus, which operates according to the following principle: when the method is used, a programming program is used for defining the required operation steps, and the automatic operation state can be started after the command is confirmed. And the reagent is added and the sample is transferred by fixing the pipette head through an automatic mechanical arm. After the mixed liquid is transferred to the oscillator module, the order triggers the vibration mode afterwards, accomplishes the mixture of sample, carries out the heating enzymolysis process afterwards, and the extraction process of whole glucocorticoid all can be accomplished through automatic instrument, improves the convenience of experiment, mechanical operation is convenient, the result is stable, and the repeatability is high.
Example 2
The embodiment 2 of the present invention provides a method for detecting endogenous glucocorticoid, which is similar to the embodiment 1, except that the pH of the phosphate buffer is 7.5.
The cortisone standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.98725, LOD 0.1ng/mL, LOQ 1.5 ng/mL.
Example 3
Example 3 of the present invention provides a method for detecting endogenous glucocorticoid, which is similar to example 1, except that the amount of methyl tert-butyl ether added is 0.
The cortisone standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.81275, LOD 1ng/mL, LOQ 5 ng/mL.
Example 4
The embodiment 4 of the present invention provides a method for detecting endogenous glucocorticoid, which is similar to the embodiment 1, but the difference is that the chromatographic column is a reversed-direction C18 chromatographic column.
The cortisone standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.76418, LOD 2ng/mL, LOQ 5 ng/mL.
Example 5
The embodiment 5 of the invention provides a method for detecting endogenous glucocorticoid, which is the same as the embodiment 1 except that the pH of the alkali liquor is 10.5.
The cortisone standard curve drawing, detection limit and quantitative limit testing method is the same as example 1, and R is obtained20.83147, LOD 3ng/mL, LOQ 6 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 detecting endogenous glucocorticoid, which is characterized by comprising the following steps:
(1) extraction of glucocorticoids: putting the biological liquid into a container, adding an internal standard substance, an acidifying reagent and enzyme, and carrying out enzymolysis for 1-3h at 50-60 ℃; adding alkali liquor and ether substances after enzymolysis, shaking, centrifuging, collecting supernatant, and adding N2Drying, adding the extracting solution for redissolving to obtain a solution to be detected;
(2) and (4) carrying out liquid chromatography and mass spectrum detection on the liquid to be detected, and analyzing to obtain the product.
2. The method for detecting endogenous glucocorticoid according to claim 1, wherein the alkaline solution is one or more selected from the group consisting of sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate.
3. The method for detecting endogenous glucocorticoid according to claim 2, wherein the alkaline solution is sodium bicarbonate or sodium carbonate, and the pH of the alkaline solution is 9 to 10.
4. The method for detecting an endogenous glucocorticoid according to claim 3, wherein the ether substance has 4 to 8 carbon atoms.
5. The method for detecting endogenous glucocorticoid according to claim 4, wherein the volume ratio of the ether substance to the alkaline solution is (70-90): 1.
6. the method for detecting an endogenous glucocorticoid according to any one of claims 1 to 5, wherein the volume ratio of the biological fluid to the internal standard substance is (8-12): 1.
7. the method for detecting an endogenous glucocorticoid according to claim 6, wherein the extraction liquid is water or acetonitrile.
8. The method for detecting an endogenous glucocorticoid according to claim 7, wherein the volume ratio of the water to the acetonitrile is (2-6): 1.
9. the method for detecting endogenous glucocorticosteroids according to claim 7 or 8, wherein the liquid chromatography column is selected from any one of reversed-phase C18, T3 and F5 columns in the liquid chromatography detection.
10. Use of the method according to any one of claims 1-9 for the quantitative detection of endogenous glucocorticoids in urine, saliva or plasma.
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| J. PITARCH-MOTELLÓN等: "Determination of selected endogenous anabolic androgenic steroids and ratios in urine by ultra high performance liquid chromatographytandem mass spectrometry and isotope pattern deconvolution", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 杨树民: "兴奋剂药物的液质联用检测方法", 《基础医学与临床》 * |
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