CN114689734A - A method for detecting the origin of steroid steroids and its application - Google Patents

Abstract

The invention belongs to the technical field of extraction and analytical chemistry, and particularly relates to a steroid substance source detection method and application thereof, wherein the steroid substance source detection method comprises the following steps: GC-C-IRMS detection and analysis delta is carried out after steroid substances in the sample are extracted¹³And (4) C value. The GC-C-IRMS detection method has the detection concentration linear range of 10-13000 ng/mL, and when a 3mL urine sample is taken, the lowest detection concentration of T and 5 alpha-diol with lower contents in the urine sample is 10ng/mL, and the lowest detection concentration of An and Etio with higher concentrations in the urine sample is 100 ng/mL. The detection process only needs one-time liquid phase separation and does not need derivatization; mass spectrometric detectionThe process instrument has good linearity, independent linear model test is carried out on each analyte, the obtained uncertainty result accords with the latest WADA2021 detection standard, the analysis time is short, the efficiency is high, and the requirement of the excitation agent detection of the large-scale events is met.

Description

A method for detecting the origin of steroid steroids and its application

technical field

The invention belongs to the technical field of extraction and its analytical chemistry, and more particularly, the invention relates to a method for detecting the origin of steroid steroids and its application.

Background technique

Endogenous steroids can regulate the level of metabolism in the body, so anabolic steroids based on this are listed as prohibited drugs by the International Olympic Committee. At the same time, due to the complex and changeable forms of such substances in human urine, it is not easy to detect, such as testosterone, because the molecular structure of testosterone synthesized in the body and testosterone in testosterone preparations is completely consistent, conventional liquid/gas mass spectrometers cannot Distinguish between the body's own synthetic testosterone and exogenously ingested testosterone. At the same time, due to individual differences and different physiological conditions, the concentration of testosterone in urine samples varies widely. Therefore, the confirmation of exogenous testosterone and its metabolites in urine samples has always been the focus and difficulty in the field of doping testing. . The establishment of a method for the detection of steroid sources in urine by isotope ratio mass spectrometry has become the top priority of doping testing, and it is of great significance to determine whether endogenous steroid stimulants are used.

At present, the research on the detection of endogenous steroids by isotope ratio mass spectrometry has not gone through a long time. In 1990, it was first reported that the δ ¹³ C value of testosterone secreted by the human body is different from that of synthetic testosterone, which proves that the analytical method is different. It has great application value in doping detection. Most synthetic steroid hormones are derived from plant substrates, and synthetic steroid hormones have poorer δ ¹³ C values than the body's own synthetic steroid hormones. When the human body ingests exogenous hormones, the δ ¹³ C value of endogenous substances and their metabolites in the body will change accordingly, while other steroid hormones unrelated to the metabolism of exogenous hormones will not be affected. At the same time, the methods of sample pretreatment in different countries are quite different, and each has its own characteristics. At present, the commonly used method in the world is to use two liquid phase separation and purification to improve the purity of the analyte, or to chemically derivatize the analyte. completely separated. However, the common problems to be solved are: tedious operation, time-consuming, low sensitivity, and large sample consumption.

SUMMARY OF THE INVENTION

In view of some problems existing in the prior art, a first aspect of the present invention provides a method for detecting the origin of steroids, including: GC-C-IRMS detection after extracting steroids in a sample The δ ¹³ C values were analyzed.

As a preferred technical solution of the present invention, the GC-C-IRMS detection and analysis process includes: gas chromatographic analysis, temperature-increasing combustion program and isotope ratio mass spectrometry analysis of steroid sterols, respectively, wherein the chromatographic analysis in gas chromatographic analysis The heating program of the column includes: heating to 150°C, holding for 1-5min; heating to 200°C at 30°C/min; then heating to 200-290°C at 2.5°C/min, holding for 1-3min; then heating at 30°C/min The temperature was raised to 290-350°C and kept for 1-3min.

As a preferred technical solution of the present invention, in the gas chromatography analysis, the column length of the chromatographic column is 15-60 m, the inner diameter is 0.25-0.32 mm, and the liquid film thickness is 0.15-0.25 μm.

As a preferred technical solution of the present invention, the process of extracting steroids in the sample includes: under the action of buffer and enzyme, the sample is enzymatically hydrolyzed at 50-60°C for 1-3 hours, cooled, and added to The lye and extractant were shaken and centrifuged, and the supernatant was taken, dried with N at 65-75 °C, and then added with high performance liquid chromatography eluate for HPLC separation and purification _; then dried at 65-80 °C with _N2 , Use the IRMS on-machine solution to dissolve, that is, it is obtained.

As a preferred technical solution of the present invention, the buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer solution is 6-6.9.

As a preferred technical solution of the present invention, the concentration of the alkaline solution is 15-25 wt%.

As a preferred technical solution of the present invention, the solute in the alkaline solution is sodium carbonate and/or sodium bicarbonate.

As a preferred technical solution of the present invention, the IRMS on-machine solution includes organic esters and/or C4-C8 alkanes.

As a preferred technical solution of the present invention, the extraction agent is an organic ether solvent with a weight average molecular weight of 70-100.

The second aspect of the present invention provides an application of the method for detecting the origin of steroids in detecting stimulants in urine.

Compared with the prior art, the present invention has the following advantages:

(1) Using the method for detecting the origin of steroids in the present application, especially after extracting the steroids in the urine according to the method of the present application, GC-C-IRMS detection and analysis can be performed, which can be 100% accurately determined. Is it endogenous or exogenous;

(2) the application adopts methyl tertiary butyl ether as the extraction agent, the extraction is complete, and the detection limit is low, which increases the applicability of the detection method of the application;

(3) In this application, when the pH of the phosphate buffer is controlled at 6-7, especially 6.86, after the enzymatic hydrolysis in the later stage of the application, the judgment of steroid steroids after the GC/C/IRMS detection of the application is more accurate. to be accurate;

(4) The linear range of the detection concentration of the GC-C-IRMS detection method established by the present invention is 10-13000ng/mL, and the limit of quantification is as low as 10ng/mL. During the detection process, it is not affected by different constitutions, ages, races and sports items The restrictions apply to a wide range;

(5) The detection method developed by the present invention has good stability and meets the uncertainty detection standard of WADA for endogenous steroid stimulants;

(6) Compared with the commonly used detection method of the combination of secondary liquid phase separation and chemical derivatization, in this application, only one liquid phase separation is required, no derivatization is required, the analysis time is short, and the detection efficiency is high.

Description of drawings

Figures 1-8 are the linear distribution diagrams of Etio, An, PD, T, 5βdiol, 5αdiol, ET, and 11OHAn;

Figures 9-15 are the linear fitting models of Keeling plot;

Fig. 16 is the isotope spectrum obtained by the sample injection detection standard mixture (5α-diol, 5β-diol, ET, 11OHAn) of the present invention;

Fig. 17 is the isotope spectrum obtained by the sample injection detection standard mixture (Etio, An, PD, T) of the present invention;

Fig. 18 is the isotope spectrum obtained by injecting and detecting a single substance (11OHAn) in the sample according to the present invention;

Fig. 19 is the isotope spectrum obtained by injecting and detecting a single substance (T) in the sample according to the present invention;

Fig. 20 is the isotope spectrum obtained by injecting and detecting a single substance (5β-diol) in the sample according to the present invention;

Fig. 21 is the isotope spectrum obtained by injecting and detecting a single substance (5α-diol) in the sample according to the present invention;

Fig. 22 is the isotope spectrum obtained by injecting and detecting a single substance (Etio) in the sample according to the present invention;

Fig. 23 is the isotope spectrum obtained by injecting and detecting a single substance (An) in the sample according to the present invention;

Fig. 24 is the isotope spectrum obtained by injecting and detecting a single substance (PD) in the sample according to the present invention

Detailed ways

The content of the present invention may be more readily understood by reference to the following detailed description of the preferred embodiments of the invention and the included examples. Unless otherwise defined, 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 definitions in this specification will control.

The first aspect of the present invention provides a method for detecting the origin of steroid steroids. After extracting the steroid steroids in a sample, GC-C-IRMS is performed to detect and analyze the δ ¹³ C value.

Preferably, the GC-C-IRMS conditions are:

The column temperature adopts programmed temperature rise: the injection volume is 1-4ul, carbon dioxide is used as the reference gas, helium is used as the carrier gas, and the combustion furnace temperature is 950-1050℃.

In one embodiment, the GC-C-IRMS detection and analysis process includes: gas chromatography analysis, temperature-increasing combustion program and isotope ratio mass spectrometry analysis for steroid steroids, wherein the temperature program of the chromatographic column in the gas chromatography analysis Including: heating to 150°C, holding for 1-5min; heating to 200°C at 30°C/min; then heating to 200-290°C at 2.5°C/min, holding for 1-3min; then heating to 290-290°C at 30°C/min 350 ℃, keep warm for 1-3min.

Preferably, in the gas chromatographic analysis, the chromatographic column adopts temperature programming: 150°C (1min)-30°C/min→270°C-2.5°C/min→290°C (1.5min)-30°C/min→300°C (1.5°C) min). .

Endogenous and exogenous steroid steroid substances cannot be judged in the prior art, resulting in limited detection of steroid steroid stimulants. After a series of studies in the experiment, the applicant unexpectedly found It was found that when the steroid steroids in the urine obtained in this application were analyzed by GC-C-IRMS according to the specific procedure in this application, the exogenous and endogenous steroids in human urine could be distinguished. Steroid substances, especially after being analyzed by the gas chromatographic column specified in the application, and then heated by the combustion program of the specific degree of the application, can make the endogenous and exogenous steroid steroid substances in the urine. Judgment reaches 100% accuracy. The applicant speculates that the molecular structure of steroid steroids synthesized by the human body may be different from that of exogenous steroids. Analysis, according to the measured δ ¹³ C value, after eliminating the interference of other C-containing substances in the urine, the analyzed δ ¹³ C value at this time can accurately determine whether it contains exogenous steroid steroids.

In one embodiment, in the gas chromatography analysis, the column length of the chromatographic column is 15-60 m, the inner diameter is 0.25-0.32 mm, and the liquid film thickness is 0.15-0.25 μm.

Preferably, the type of the chromatographic column in the gas chromatographic analysis is a DB-17 capillary chromatographic column.

In one embodiment, the process of extracting steroids in the sample includes: under the action of a buffer and an enzyme, the sample is hydrolyzed at 50-60° C. for 1-3 hours to obtain an enzymatic hydrolysis solution, cooled, Add lye and extractant, shake and centrifuge, take the supernatant, blow dry at 65-75°C with N ₂ and add high performance liquid chromatography eluent for HPLC separation and purification _; then blow dry at 65-80° C. , use the IRMS on-machine solution to dissolve, that is, it is obtained.

In a preferred embodiment, the process of extracting steroids in the sample includes: under the action of a buffer and an enzyme, the sample is enzymatically hydrolyzed at 50-60° C. for 1-3 hours to obtain an enzymatic hydrolysis solution, Cool, add lye and extractant, shake at 2000-3000rpm for 1-3min, centrifuge at 2500-3500rpm at 0-10°C for 3-5min, and then freeze in -20°C ethanol solution for 3-5min, Take the supernatant, dry it with N at 65-75 °C, and add high performance liquid chromatography eluent for HPLC separation and purification; then dry it at 65-80 °C with N, use the IRMS _{on -} machine solution to dissolve, namely have to.

In a further preferred embodiment, the process of extracting steroids in the sample includes: under the action of a buffer and an enzyme, the sample is enzymatically hydrolyzed at 55°C for 2 hours to obtain an enzymatic hydrolysis solution, cooled to room temperature, and added to The lye and extractant were shaken at 2500 rpm for 3 min, centrifuged at 3000 rpm at 4 °C for 3 min, and then frozen in -20 °C ethanol solution for ₃ min. Cool to room temperature, redissolve with eluent (the volume ratio of methanol, water and acetonitrile is 5:4:1 mixture, 55ul), add high performance liquid chromatography eluent for HPLC separation and purification; then at 75 ° C N ₂ After drying, it was cooled to room temperature and dissolved with IRMS on-machine solution.

The sample described in this application is urine.

Preferably, the buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the buffer solution is 6-6.9; more preferably, the buffer solution is sodium dihydrogen phosphate and disodium hydrogen phosphate The pH of the phosphate buffer is 6.86.

Preferably, the volume ratio of the buffer to urine is 1:(1-3); more preferably, the volume ratio of the buffer to urine is 1:2.

The enzymes of the present invention are not particularly limited, and those skilled in the art can make routine selections.

Preferably, the enzyme is glucuronidase.

Preferably, the volume ratio of the sample to the enzyme is (20-50):1; more preferably, the volume ratio of the sample to the enzyme is 40:1.

Preferably, the concentration of the alkali solution is 15-25wt%; more preferably, the concentration of the alkali solution is 20wt%.

Preferably, the solute in the lye solution is sodium carbonate and/or sodium bicarbonate.

In this application, the solute of the alkaline solution is not particularly limited, and those skilled in the art can make routine selections according to the description in this application.

Preferably, the volume ratio of the enzymatic hydrolysis solution and the alkaline solution is (25-40): 1; more preferably, the volume ratio of the enzymatic hydrolysis solution and the alkaline solution is 35:1.

Preferably, the extractant is an organic ether solvent with a weight average molecular weight of 70-100; more preferably, the extractant is methyl tert-butyl ether.

Preferably, the volume ratio of the enzymatic hydrolysis solution and the extractant is (1-1.5):1; more preferably, the volume ratio of the enzymolysis solution and the extractant is 1.2:1.

Preferably, the HPLC eluent is methanol solution or a mixed solution of methanol, water and acetonitrile.

Preferably, the volume ratio of methanol, water and acetonitrile in the HPLC eluent is 5:4:1.

Preferably, the high performance liquid chromatography eluate contains 0.3 mg/mL of the internal standard methyl testis.

The addition amount of the HPLC eluent described in this application is not particularly limited, and those skilled in the art can make routine selections.

Preferably, the IRMS running solution includes organic esters and/or C4-C8 alkanes; further preferably, the IRMS running solution includes ethyl acetate and n-hexane.

Preferably, the volume ratio of the ethyl acetate to n-hexane is (1-5):1; more preferably, the volume ratio of the ethyl acetate to n-hexane is 3:1.

In this application, the added amount of the IRMS on-machine solution is not particularly limited, and those skilled in the art can make routine selections according to the description in this application.

In one embodiment, the condition of described HPLC separation and purification comprises:

Chromatographic column: C18 chromatographic column; mobile phase: water: acetonitrile (gradient elution water: acetonitrile = 60:40, 50:50 in 2min, retention 23min, 10:90 in 5min, return to initial mobile phase 60 in 1min : 40, retention 5min); flow rate: 1mL/min; injection volume: 50uL; column temperature: 38°C.

In one embodiment, the chromatographic column for separation and purification by HPLC is selected from any one of reverse-phase C18, T3, and F5 chromatographic columns.

Preferably, the chromatographic column for separation and purification by HPLC is a C18 chromatographic column.

In one embodiment, the mobile phases are water and acetonitrile during the purification of the gas chromatography analysis chromatographic column.

Preferably, in the purification of the gas chromatography column, the volume ratio of water and acetonitrile in the mobile phase is (50-70): 40; more preferably, in the purification of the gas chromatography column, water and acetonitrile in the mobile phase are The volume ratio of acetonitrile was 60:40.

In one embodiment, during the HPLC separation and purification, the flow rate of the mobile phase is 0.5-1.5 mL/min.

Preferably, during the HPLC separation and purification, the flow rate of the mobile phase is 1 mL/min.

In one embodiment, during the HPLC separation and purification, the column temperature of the chromatographic column is 10-45°C.

Preferably, during the HPLC separation and purification, the column temperature of the chromatographic column is 38°C.

In one embodiment, during the HPLC separation and purification, the chromatographic column purification is 100% isocratic elution, and the elution time is 10-40 min.

Preferably, the elution time is 36 min.

In one embodiment, the GC/IRMS conditions are:

Chromatographic column: DB-17column, inlet temperature: 280 °C, interface temperature: 300 °C, constant flow mode, injection 1uL.

Mass spectrometry ion source: EI, 70eV, acquisition mode: SCAN mode, acquisition mass range: 50-500amu.

The second aspect of the present invention provides an application of the method for detecting the origin of steroids in detecting stimulants in urine.

Example

Hereinafter, the present invention will be described in more detail by means of examples, but it should be understood that these examples are merely illustrative and not restrictive. Unless otherwise stated, the raw materials used in the following examples are all commercially available.

Example

An embodiment of the present invention provides a method for detecting the origin of steroid steroids, which is specifically as follows:

Add 3 mL of urine to an 8 mL glass test tube, then add 1.5 mL of phosphate buffered saline (PBS) and 75 uL of glucuronidase, mix well, and enzymatically hydrolyze at 55°C for 2 hours in a water bath; after enzymatic hydrolysis, cool to room temperature , add 150uL of 20wt% sodium carbonate and 4mL of methyl tertiary butyl ether (MTBE), shake at 2500rpm for 3min, then centrifuge at 4°C and 3000rpm for 3min, after centrifugation, put it in -20°C ethanol solution for freezing After 3 min, the upper organic phase was transferred to a clean test tube and dried under _N2 at 65 °C. After cooling to room temperature, it was reconstituted with 55 ul of eluent (a mixed solution of methanol, water and acetonitrile in a volume ratio of 5:4:1), shaken, and transferred to a liquid injection vial for separation and purification by HPLC. Wait for HPLC to separate the components, blow dry the collected components under N at 75°C, cool to room temperature, and reconstitute them with the IRMS liquid _on the machine (ethyl acetate: n-hexane volume ratio of 1:3). Mixed solution, 20ul), carry out GC-C-IRMS detection; Described phosphate buffer is the mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and pH is 6.86; Described IRMS upper machine fluid is that volume ratio is 3: 1 of ethyl acetate and n-hexane.

Wherein, the HPLC separation conditions are as follows:

Chromatographic column: C18 chromatographic column; mobile phase: water: acetonitrile (gradient elution water: acetonitrile = 60:40, 50:50 in 2min, retention 23min, 10:90 in 5min, return to initial mobile phase 60 in 1min : 40, retention 5min); flow rate: 1mL/min; injection volume: 50uL; column temperature: 38°C.

Among them, the detection conditions of GC-C-IRMS are as follows: Column: DB-17 capillary column, (30mX0.25umi.d.X0.25 um thickness); inlet temperature: 280℃, interface temperature: 300℃, constant temperature Flow mode, inject 1uL.

In the gas chromatography analysis, the column temperature of the chromatographic column adopts a programmed temperature rise: 150℃(1min)-30℃/min→270℃-2.5℃/min→290℃(1.5min)-30℃/min→300℃(1.5min). Furnace temperature: 1000°C, acceleration voltage: 3KeV.

Mass spectrometry ion source: EI, 70eV, acquisition mode: SCAN mode, acquisition mass range: 50-500amu.

Figure 16 is the isotope spectrum obtained by the sample injection detection standard mixture (5α-diol, 5β-diol, ET, 11OHAn) of the present invention; Figure 17 is the sample injection detection standard mixture (Etio, An, PD, T) obtained isotope spectrum; Fig. 18 is the isotope spectrum obtained by injecting a single substance (11OHAn) in the sample of the present invention; Fig. 19 is the isotope spectrum obtained by injecting and detecting a single substance (T) in the sample of the present invention; Fig. 20 is the isotope spectrum obtained by a single substance (5β-diol) in the sample injected and detected by the present invention; Fig. 21 is the isotope spectrum obtained by a single substance (5α-diol) in the sample by the present invention; Fig. 22 is the The isotope spectrum of a single substance (Etio) in the sample of the present invention is injected and detected; FIG. 23 is the isotope spectrum of a single substance (An) obtained by the sample of the present invention by the injection of the sample; FIG. 24 is the single substance of the sample of the present invention. (PD) Obtained isotope spectra.

Instrument Linearity:

According to the requirements of international technical documents, the instrument linearity evaluation of GC-C-IRMS method requires that the standard deviation (SD) of the δ ¹³ C value of the substances measured in parallel must be less than 0.5. In this research method, we formulated each standard into 6 solutions of different concentrations (Etio, An, PD, T, 5βdiol, 5αdiol, ET, 11OHAn, 10, 20, 50, 80, 150, 200 ng, respectively /ul of single standard solution), each solution was injected with 0.5-3.0 μL, and the test was tested in parallel three times. A series of [delta] ^13C values were obtained for each analyte at different signals. Figures 1-8 are the linear distributions of Etio, An, PD, T, 5βdiol, 5αdiol, ET, and 11OHAn respectively; Table 1 lists the measurement results of the linearity of the instrument using standard substances. According to the obtained measurement results, all the detected substances have stable δ ¹³ C values at 147-5625 mV. Therefore, in the actual detection, it is necessary to concentrate or dilute the processed sample before it is put on the machine to obtain an appropriate concentration, and to ensure that the sample signal is within the linear range of the instrument to obtain an accurate value.

Table 1

The lowest detection concentration in this study method is the lowest concentration of the test substance determined based on a 3 mL urine sample. Usually An and Etio are at higher concentrations in urine samples, so this method is only validated for pure concentrations of 100ng/mL urine samples, PD and 11OHAn for 50ng/mL urine samples, 5β-diol for 20ng/mL urine samples, other The ERC validated a 10ng/mL urine sample. During the validation process, three assays were performed on three consecutive days, and three results were obtained for the same analyte. From the results in Table 2 and Table 3, when 3 mL of urine sample is used for analysis, stable δ13C values can be measured for T and 5α-diol with a content of 10 ng/mL using this method.

Table 2

table 3

Uncertainty Test:

The combined uncertainty of the method in this paper includes the rms mean square of method intermediate precision and bias. The specific formula is as follows:

where Uc is the uncertainty of the method, Sw is the contribution to the intermediate precision of the method, and RMSbias is the contribution of the method measurement bias. In actual determination, Sw is the standard deviation of the measurement results of negative control urine samples and positive quality control urine samples. The method measurement deviation is obtained by the linear fitting result of Keeling plot. Table 4 lists the evaluation results of uncertainty. It can be seen from the table that the Sw of the measured quality control samples are between 0.2 and 0.6 respectively, indicating that this method has high stability, and all the measured uncertainties meet the WADA requirements. Uncertainty testing requirements for specific steroid stimulant substances. Figures 9-15 are the obtained Keeling plot linear fitting models, which are used for method measurement deviation calculation.

Table 4

PD 11OHAn T 5β-diol 5α-diol Etio An Intermediate Precision(‰) 0.2 0.3 0.57 0.56 0.3 0.2 0.47 Biasδ¹³C(‰) 0.2 0.004 0.5 0.1 0.5 0.3 0.2 u_c(‰) 0.3 0.3 0.758 0.6 0.6 0.4 0.5

The foregoing examples are illustrative only and serve to explain some of the features of the methods described herein. The appended claims are intended to claim the broadest conceivable scope and the embodiments presented herein are merely illustrative of selected implementations according to a combination of all possible embodiments. Accordingly, it is the applicant's intention that the appended claims not be limited by the selection of examples that characterize the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be construed, where possible, to be covered by the appended claims.

Claims (10)

1. A method for detecting the source of steroid substances is characterized by comprising the following steps: GC-C-IRMS detection and analysis delta is carried out after steroid substances in the sample are extracted¹³And C value.

2. The method for detecting the provenance of steroidal substances according to claim 1, wherein the GC-C-IRMS detection analysis process comprises: the steroid substance is subjected to gas chromatography, a heating combustion program and isotope ratio mass spectrometry respectively, wherein the heating program of a chromatographic column in the gas chromatography comprises the following steps: heating to 150 deg.C, and maintaining for 1-5 min; heating to 200 deg.C at 30 deg.C/min; then heating to 200 and 290 ℃ at the speed of 2.5 ℃/min, and preserving the heat for 1-3 min; then raising the temperature to 350 ℃ at the speed of 30 ℃/min, and preserving the heat for 1-3 min.

3. The method for detecting the provenance of steroidal substances according to claim 2, wherein said gas chromatography column has a column length of 15 to 60m, an inner diameter of 0.25 to 0.32mm, and a liquid membrane thickness of 0.15 to 0.25 μm.

4. The method for the endogenous detection of steroidal substances according to any one of claims 1 to 3, wherein said process for extracting steroidal substances from a sample comprises: subjecting the sample to enzymolysis at 50-60 deg.C for 1-3 hr under the action of buffer solution and enzyme, cooling, adding alkali solution and extractant, shaking, centrifuging, and collecting supernatantLiquid at 65-75 ℃ N₂Drying, and adding high performance liquid chromatography eluent to perform HPLC separation and purification; then at 65-80 ℃ N₂And after drying, dissolving by using IRMS machine solution to obtain the product.

5. The method for the endogenous detection of steroidal substances according to claim 4, wherein said buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and said phosphate buffer has a pH of 6 to 6.9.

6. The method according to claim 5, wherein the concentration of the alkaline solution is 15 to 25 wt%.

7. The method according to claim 6, wherein the solute in the alkaline solution is sodium carbonate and/or sodium bicarbonate.

8. The method for the exogenous detection of steroid substances according to any one of claims 5 to 7, wherein the IRMS on-board solution comprises organic esters and/or C4-C8 alkanes.

9. The method of detecting the provenance of steroid substances as claimed in claim 8, wherein the extractant is an organic ether solvent having a weight average molecular weight of 70 to 100.

10. Use of a method for the endogenous detection of a steroid in accordance with any one of claims 1 to 9 for the detection of an stimulant in urine.

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