CN114689734A - Steroid substance source detection method and application thereof - Google Patents

Steroid substance source detection method and application thereof Download PDF

Info

Publication number
CN114689734A
CN114689734A CN202110347345.4A CN202110347345A CN114689734A CN 114689734 A CN114689734 A CN 114689734A CN 202110347345 A CN202110347345 A CN 202110347345A CN 114689734 A CN114689734 A CN 114689734A
Authority
CN
China
Prior art keywords
detection
steroid
sample
irms
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110347345.4A
Other languages
Chinese (zh)
Inventor
刘冰
卓悦
徐昕
陈佩杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai University of Sport
Original Assignee
Shanghai University of Sport
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai University of Sport filed Critical Shanghai University of Sport
Priority to CN202110347345.4A priority Critical patent/CN114689734A/en
Publication of CN114689734A publication Critical patent/CN114689734A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

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

Steroid substance source detection method and application thereof
Technical Field
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.
Background
Endogenous steroidal drugs can regulate in vivo metabolic levels, and synthetic steroidal drugs based on this are therefore classified as illicit drugs by the international oeuv. Meanwhile, because the substances exist in human urine in a complex and changeable form and are difficult to detect, such as testosterone, and because the molecular structures of the testosterone synthesized in vivo and the testosterone in a testosterone preparation are completely consistent, a conventional liquid phase/gas phase mass spectrometer cannot distinguish the testosterone synthesized in vivo from the testosterone ingested by an external source. Meanwhile, due to individual differences and different physiological conditions, the concentration of testosterone in the urine sample has a large variation range, so that the confirmation of exogenous testosterone and metabolites thereof in the urine sample is always the key point and difficulty in the field of stimulant detection. The method for establishing isotope ratio mass spectrometry to detect the source of the steroid in urine is the important factor for detecting the stimulant, and has important significance for determining whether the endogenous steroid stimulant is used.
Currently, studies using isotope ratio mass spectrometry to detect endogenous steroids have not been long-lasting, and delta of testosterone secreted by the human body itself and testosterone in a synthetic formulation was first reported in 199013The C values are different, and the analysis method is proved to have great application value in the stimulant detection. Most synthetic steroid hormones are derived from plant substrates and have a much lower delta than the human body's own synthetic steroid hormones13And C value. After the human body takes in exogenous hormone, the delta of endogenous substances and metabolites thereof in the human body13The C value will change, while other steroid hormones not involved in the metabolism of exogenous hormones are not affected. Meanwhile, the methods for pretreatment of samples in various countries have great difference and various characteristics, and the current international common method is to use two times of liquid phase separation and purification to improve the purity of the analyte or to completely separate the analyte on a gas chromatograph by a chemical derivatization means. But the problems to be solved in common are: complicated operation, time consumption, low sensitivity and simple operationThe method developed by the invention is relatively quick, has good reproducibility and less sample consumption, and meets the sample analysis requirement.
Disclosure of Invention
In view of the problems in the prior art, the first aspect of the present invention provides a method for detecting the source of steroidal substances, comprising: GC-C-IRMS detection and analysis delta is carried out after steroid substances in the sample are extracted13And (4) C value.
As a preferred technical scheme of the invention, the GC-C-IRMS detection and analysis process comprises the following steps: 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.
As a preferable technical scheme of the invention, the column length of the chromatographic column in the gas chromatographic analysis is 15-60m, the inner diameter is 0.25-0.32mm, and the liquid film thickness is 0.15-0.25 μm.
As a preferred technical solution of the present invention, the process for extracting steroid substances in 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 for centrifugation, collecting supernatant, and subjecting to N treatment at 65-75 deg.C2Drying, 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.
In a preferred embodiment of the present invention, the buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 6 to 6.9.
As a preferred technical scheme of the invention, the concentration of the alkali liquor is 15-25 wt%.
As a preferable technical scheme of the invention, the solute in the alkali liquor is sodium carbonate and/or sodium bicarbonate.
As a preferred technical scheme of the invention, the IRMS on-board solution comprises organic esters and/or C4-C8 alkane.
In a preferred embodiment of the present invention, the extractant is an organic ether solvent having a weight average molecular weight of 70 to 100.
The invention provides an application of the steroid substance source detection method in detecting stimulant in urine.
Compared with the prior art, the invention has the following advantages:
(1) by adopting the steroid substance source detection method, especially by performing GC-C-IRMS detection analysis after the steroid substances in urine are extracted according to the method, whether the steroid substances are endogenous or exogenous can be accurately judged by 100%;
(2) according to the method, the methyl tert-butyl ether is used as an extracting agent, so that the extraction is complete, the detection limit is low, and the applicability of the detection method is improved;
(3) when the pH value of the phosphate buffer solution is controlled to be 6-7, particularly 6.86, after the later enzymolysis, the judgment on steroid substances is more accurate after GC/C/IRMS detection;
(4) the GC-C-IRMS detection method established by the invention has the advantages that the linear range of the detection concentration is 10-13000 ng/mL, the quantitative limit is as low as 10ng/mL, the detection process is not limited by different constitutions, ages, ethnicities and sports items, and the application range is wide;
(5) the detection method developed by the invention has good stability and meets the uncertainty detection standard of WADA for endogenous steroid stimulant;
(6) compared with the international commonly used detection method combining secondary liquid phase separation and chemical derivatization, only one-time liquid phase separation is needed in the method, derivatization is not needed, the analysis time is short, and the detection efficiency is high.
Drawings
FIGS. 1-8 are linear profiles of Etio, An, PD, T, 5. beta. diol, 5. alpha. diol, ET, 11OHAN, respectively;
FIGS. 9-15 are graphs of a linear fit model for Keeling plot, respectively;
FIG. 16 is an isotope diagram obtained by sample injection detection of a standard mixture (5. alpha. -diol, 5. beta. -diol, ET, 11OHAN) according to the present invention;
FIG. 17 is An isotope diagram obtained by sample injection detection of mixed standard (Etio, An, PD, T) in accordance with the present invention;
FIG. 18 is an isotope diagram of a single substance (11OHAN) detected in a sample by a sample injection method according to the present invention;
FIG. 19 is an isotope diagram of a single substance (T) in a sample detected by the present invention;
FIG. 20 is an isotope diagram of a single substance (5. beta. -diol) detected in a sample by sample injection according to the present invention;
FIG. 21 is an isotope diagram of a single substance (5 α -diol) in a sample detected by the present invention;
FIG. 22 is an isotope diagram of the present invention taken in a sample to detect a single substance (Etio);
FIG. 23 is An isotope diagram of a single substance (An) in a sample detected by the present invention;
FIG. 24 is an isotope diagram of a single substance (PD) in a sample detected by injection according to 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 detecting the source of steroid substances, which comprises the steps of extracting steroid substances in a sample, and then carrying out GC-C-IRMS (gas chromatography-tandem mass spectrometry) detection analysis delta13And C value.
Preferably, the GC-C-IRMS conditions are:
column temperature adopts programmed temperature rise: the sample introduction volume is 1-4ul, carbon dioxide is used as reference gas, helium is used as carrier gas, and the temperature of a combustion furnace is as follows: 950 ℃ and 1050 ℃.
In one embodiment, 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.
Preferably, the chromatographic column in the gas chromatographic analysis adopts programmed temperature rise: 150 deg.C (1min) -30 deg.C/min → 270 deg.C-2.5 deg.C/min → 290 deg.C (1.5min) -30 deg.C/min → 300 deg.C (1.5 min). .
The applicant has surprisingly found that, after a series of researches in experiments, the steroid substances in urine obtained in the application are subjected to GC-C-IRMS detection and analysis by a specific program in the application, the exogenous and endogenous steroid substances in the urine of a human body can be distinguished, and particularly, after the steroid substances are subjected to specific gas chromatographic column analysis in the application, the endogenous and exogenous steroid substances in the urine can be judged to reach 100% accuracy by heating through a specific combustion program in the application. The applicant conjectures that the molecular structures of steroid substances possibly synthesized by the human body are different from those of exogenous steroid substances, and the steroid substances are extracted by a specific extraction method in the application, subjected to GC-C-IRMS detection and analysis and then subjected to delta measurement13C value, delta analyzed at this time after eliminating interference from other C-containing substances in urine13The C value can accurately obtain whether the exogenous steroid substance is contained.
In one embodiment, the column of the gas chromatography has a column length of 15 to 60m, an inner diameter of 0.25 to 0.32mm and a liquid film thickness of 0.15 to 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 the steroidal substances in the sample comprises: subjecting the sample to enzymolysis at 50-60 deg.C for 1-3 hr under the action of buffer solution and enzyme to obtainAdding the enzymolysis solution, cooling, adding alkali solution and extractant, shaking and centrifuging, collecting supernatant, and adding N at 65-75 deg.C2Drying, 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.
In a preferred embodiment, the process of extracting the steroidal substances in the sample comprises: subjecting the sample to enzymolysis at 50-60 deg.C for 1-3h under the action of buffer solution and enzyme to obtain enzymolysis solution, cooling, adding alkali solution and extraction agent, shaking at 3000rpm of 2000-5 deg.C for 1-3min, centrifuging at 3500rpm of 2500-10 deg.C for 3-5min, freezing in ethanol solution at-20 deg.C for 3-5min, collecting supernatant, and subjecting to N-hydrolysis at 65-75 deg.C2Drying, and adding high performance liquid chromatography eluent to perform HPLC separation and purification; then at 65-80 ℃ N2And after blow-drying, dissolving the solution on an IRMS machine to obtain the product.
In a further preferred embodiment, said process of extracting steroid substances from the sample comprises: subjecting the sample to enzymolysis at 55 deg.C for 2 hr under the action of buffer solution and enzyme to obtain enzymolysis solution, cooling to room temperature, adding alkali solution and extractant, shaking at 2500rpm for 3min, centrifuging at 3000rpm at 4 deg.C for 3min, freezing in ethanol solution at-20 deg.C for 3min, collecting supernatant, and subjecting to N hydrolysis at 65 deg.C2After blow-drying, cooling to room temperature, redissolving with eluent (55 ul of mixed solution of methanol, water and acetonitrile in a volume ratio of 5:4: 1), and adding high performance liquid chromatography eluent for HPLC separation and purification; then at 75 ℃ N2And after blow-drying, cooling to room temperature, and dissolving by using an IRMS machine solution to obtain the compound.
The sample herein is urine.
Preferably, the buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value of the buffer solution is 6-6.9; more preferably, the buffer is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH of the phosphate buffer is 6.86.
Preferably, the volume ratio of the buffer solution to the urine is 1: (1-3); more preferably, the volume ratio of the buffer to urine is 1: 2.
the enzyme of the present invention is not particularly limited and may be routinely selected by those skilled in the art.
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 liquor is 15-25 wt%; more preferably, the concentration of the lye is 20 wt%.
Preferably, the solute in the alkali liquor is sodium carbonate and/or sodium bicarbonate.
In the present application, the alkali solution is not particularly limited to the solute, and those skilled in the art can make routine selections according to the description in the present application.
Preferably, the volume ratio of the enzymolysis liquid to the alkali liquor is (25-40): 1; more preferably, the volume ratio of the enzymolysis liquid to the alkali liquor is 35: 1.
preferably, the extractant is an organic ether solvent with the weight-average molecular weight of 70-100; more preferably, the extractant is methyl tert-butyl ether.
Preferably, the volume ratio of the enzymolysis liquid to the extracting agent is (1-1.5): 1; more preferably, the volume ratio of the enzymolysis liquid to the extracting agent is 1.2: 1.
preferably, the eluent of the high performance liquid chromatography is methanol solution or mixed solution of methanol, water and acetonitrile.
Preferably, the volume ratio of methanol, water and acetonitrile in the eluent of the high performance liquid chromatography is 5:4: 1.
Preferably, the eluent of the high performance liquid chromatography contains 0.3mg/mL of internal standard methyltestosterone.
The amount of the eluent for high performance liquid chromatography as described herein is not particularly limited and may be routinely selected by those skilled in the art.
Preferably, the IRMS on-board solution comprises organic esters and/or C4-C8 alkanes; further preferably, the IRMS on-machine solution comprises ethyl acetate and n-hexane.
Preferably, the volume ratio of the ethyl acetate to the n-hexane is (1-5): 1; more preferably, the volume ratio of the ethyl acetate to the n-hexane is 3: 1.
the amount of IRMS solution added in the present application is not particularly limited and may be routinely selected by those skilled in the art based on the description herein.
In one embodiment, the conditions for HPLC separation and purification include:
a chromatographic column: a C18 chromatography column; mobile phase: water: acetonitrile (gradient shower water: acetonitrile 60:40, 50:50 within 2min, retention 23min, 10:90 within 5min, return to initial mobile phase 60:40 within 1min, retention 5 min); flow rate: 1 mL/min; sample introduction amount: 50 uL; column temperature: at 38 ℃.
In one embodiment, the HPLC separation and purification column is selected from any one of reverse phase C18, T3, F5 columns.
Preferably, the chromatographic column for HPLC separation and purification is a C18 chromatographic column.
In one embodiment, the mobile phase is water and acetonitrile when the gas chromatography column is used for purification.
Preferably, the volume ratio of water to acetonitrile in the mobile phase during the purification of the gas chromatography column is (50-70): 40; more preferably, the gas chromatography column is used for purification, and the volume ratio of water to acetonitrile in the mobile phase is 60: 40.
in one embodiment, the flow rate of the mobile phase during HPLC separation and purification is 0.5-1.5 mL/min.
Preferably, the flow rate of the mobile phase is 1mL/min during the HPLC separation and purification.
In one embodiment, the HPLC separation purification is performed at a column temperature of 10-45 ℃.
Preferably, the column temperature of the chromatographic column is 38 ℃ during the HPLC separation and purification.
In one embodiment, the HPLC separation purification is performed by using a chromatographic column with 100% isocratic elution and 10-40min of elution time.
Preferably, the elution time is 36 min.
In one embodiment, the GC/IRMS conditions are:
DB-17column, injection port temperature: 280 ℃, interface temperature: 300 ℃ in constant flow mode, 1uL of sample is introduced.
Mass spectrometry ion source: EI, 70eV, acquisition mode: SCAN mode, acquisition mass range: 50-500 amu.
The invention provides an application of the steroid substance source detection method in detecting stimulant in urine.
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.
Examples
The embodiment of the invention provides a steroid substance source detection method, which comprises the following steps:
adding 3mL of urine into an 8mL glass test tube, then adding 1.5mL of Phosphate Buffered Saline (PBS) and 75uL of glucuronidase, uniformly mixing, and carrying out enzymolysis for 2h at 55 ℃ under the condition of water bath; cooling to room temperature, adding 150uL 20 wt% sodium carbonate and 4mL methyl tert-butyl ether (MTBE), shaking at 2500rpm for 3min, centrifuging at 4 deg.C and 3000rpm for 3min, freezing in-20 deg.C ethanol solution for 3min, transferring the upper organic phase into a clean test tube, and cooling at 65 deg.C under N2And (5) drying. After cooling to room temperature, redissolving with 55ul of eluent (a mixed solution of methanol, water and acetonitrile in a volume ratio of 5:4: 1), transferring to a liquid phase sampling bottle, and separating and purifying by HPLC. After HPLC separation of fractions, the fractions were collected at 75 ℃ N2Drying, cooling to room temperature, re-dissolving with IRMS machine liquid (20 ul of mixed solution of ethyl acetate and n-hexane at volume ratio of 1: 3), and performing GC-C-IRMS detection; the phosphate buffer solution is a mixed solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value is 6.86; the IRMS organic liquid is prepared by mixing the following components in a volume ratio of 3: 1 ethyl acetate and n-hexane.
Wherein, HPLC separation conditions are as follows:
a chromatographic column: a C18 chromatography column; mobile phase: water: acetonitrile (gradient shower water: acetonitrile 60:40, 50:50 within 2min, retention 23min, 10:90 within 5min, return to initial mobile phase 60:40 within 1min, retention 5 min); flow rate: 1 mL/min; sample introduction amount: 50 uL; column temperature: at 38 ℃.
Wherein, the GC-C-IRMS detection conditions are as follows: and (3) chromatographic column: DB-17 capillary chromatography column, (30mX0.25um i.d. X0.25um thickness); sample inlet temperature: 280 ℃, interface temperature: 300 ℃ in constant flow mode, 1uL of sample is introduced.
The column temperature of the chromatographic column in the gas chromatographic analysis adopts temperature programming: 150 deg.C (1min) -30 deg.C/min → 270 deg.C-2.5 deg.C/min → 290 deg.C (1.5min) -30 deg.C/min → 300 deg.C (1.5 min). Temperature of the combustion furnace: 1000 ℃, acceleration voltage: 3 KeV.
Mass spectrometry ion source: EI, 70eV, acquisition mode: SCAN mode, acquisition mass range: 50-500 amu.
FIG. 16 is an isotope diagram obtained by sample injection detection of a standard mixture (5. alpha. -diol, 5. beta. -diol, ET, 11OHAN) according to the present invention; FIG. 17 is An isotope diagram obtained by sample injection detection of mixed standard (Etio, An, PD, T) in accordance with the present invention; FIG. 18 is an isotope diagram of a single substance (11OHAN) in a sample detected by the present invention; FIG. 19 is an isotope diagram of a single substance (T) in a sample detected by the present invention; FIG. 20 is an isotope diagram of a single substance (5. beta. -diol) detected in a sample by sample injection according to the present invention; FIG. 21 is an isotope diagram of a single substance (5 α -diol) in a sample detected by the present invention; FIG. 22 is an isotope diagram of the present invention taken in a sample to detect a single substance (Etio); FIG. 23 is An isotope diagram of a single substance (An) detected in a sample by sample injection according to the present invention; FIG. 24 is an isotope diagram of a single substance (PD) detected in a sample by sample injection according to the present invention.
Instrument linearity:
according to the requirements of international technical documents, the instrument linear evaluation of GC-C-IRMS method requires the delta of substances measured in parallel13The Standard Deviation (SD) of the C value must be less than 0.5. In the present study method, we prepared each standard in 6 different concentrations of solution (Etio, An, PD, T, 5. beta. d)iol, 5 α diol, ET, 11OHAn, 10, 20, 50, 80, 150, 200ng/ul single standard solutions were prepared, respectively), each solution was injected with 0.5-3.0 μ L, and the tests were run in triplicate. Obtaining a series of deltas for each analyte at different signals13And C value. FIGS. 1-8 are linear profiles of Etio, An, PD, T, 5. beta. diol, 5. alpha. diol, ET, 11OHAN, respectively; table 1 shows the results of the measurement of the linearity of the instrument using the standard substance. According to the obtained measurement results, all the detection substances have stable delta between 147 mV and 5625mV13And C value. Therefore, in practical detection, the processed sample needs to be concentrated or diluted before being loaded into the machine to obtain a proper concentration, and the signal of the sample is ensured to be in the linear range of the instrument so as to obtain an accurate value.
TABLE 1
Figure BDA0003001179810000081
Figure BDA0003001179810000091
The lowest detection concentration in the present study method is the minimum test substance concentration determined on the basis of a 3mL urine sample. Generally, An and Etio are present in higher concentrations in urine samples, so the method only verifies pure urine samples with a concentration of 100ng/mL, PD and 11OHA verify urine samples with a concentration of 50ng/mL, 5 beta-diol verifies urine samples with a concentration of 20ng/mL, and other ERCs verify urine samples with a concentration of 10 ng/mL. During the validation process, three determinations were performed for three consecutive days, and the same analyte gave three results. From the results in tables 2 and 3, it can be seen that stable values of Δ 13C can be determined for T, 5 α -diol at a concentration of 10ng/mL using the method when a 3mL urine sample is analyzed.
TABLE 2
Figure BDA0003001179810000092
TABLE 3
Figure BDA0003001179810000093
Figure BDA0003001179810000101
And (3) uncertainty testing:
the binding uncertainty of a method herein includes the mean precision of the method and the root mean square of the deviation. The specific formula is as follows:
Figure BDA0003001179810000102
where Uc is the uncertainty of the method, Sw is the contribution to the medium precision of the method, and RMSbias is the contribution to the measurement bias of the method. In the actual measurement, Sw is the standard deviation of the measurement results of the negative quality control urine sample and the positive quality control urine sample. And the measured deviation of the method is obtained by performing linear fitting results through Keeling plot. The results of the uncertainty evaluation are shown in table 4, and it can be seen from the table that the measured quality control samples Sw are respectively between 0.2 and 0.6, which indicates that the method has high stability, and all measured uncertainties meet the uncertainty detection requirements of WADA for specific steroid stimulant substances. FIGS. 9-15 show the resulting Keeling plot linear fit models for the method measurement bias calculations, respectively.
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
uc(‰) 0.3 0.3 0.758 0.6 0.6 0.4 0.5
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 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.
CN202110347345.4A 2021-03-31 2021-03-31 Steroid substance source detection method and application thereof Pending CN114689734A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110347345.4A CN114689734A (en) 2021-03-31 2021-03-31 Steroid substance source detection method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110347345.4A CN114689734A (en) 2021-03-31 2021-03-31 Steroid substance source detection method and application thereof

Publications (1)

Publication Number Publication Date
CN114689734A true CN114689734A (en) 2022-07-01

Family

ID=82136109

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110347345.4A Pending CN114689734A (en) 2021-03-31 2021-03-31 Steroid substance source detection method and application thereof

Country Status (1)

Country Link
CN (1) CN114689734A (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105021734A (en) * 2015-07-27 2015-11-04 王静竹 Method for detection of novel testosterone agonist on the basis of etiocholanolone in urine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105021734A (en) * 2015-07-27 2015-11-04 王静竹 Method for detection of novel testosterone agonist on the basis of etiocholanolone in urine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KANG MI LEE ET AL.: "Validation and Applications of Gas Chromatography-Combustion/isotope Ratio Mass Spectrometric Method to Control Misuse of Androgens in Human" *
官凌菊;赵君军;陈颖;孙娟;蒋英;高照;张援;: "气相色谱/燃烧炉/同位素比质谱方法检测尿液中睾酮的来源" *
温超等: "气相色谱-燃烧-同位素比质谱在检测尿样中外源性睾酮及其代谢物中的应用" *
黄鸾玉;蒙源;庞燕飞;: "超高效液相色谱法测定水产品中甲基睾酮残留物" *

Similar Documents

Publication Publication Date Title
CN109470791A (en) A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol
CN108828077B (en) Kit for simultaneously detecting capecitabine and metabolite thereof in blood plasma as well as detection method and application thereof
CN109060983A (en) A kind of method of liquid chromatography-tandem mass spectrometry detection metanephrine substance
Boxler et al. Analytical considerations for (un)‐targeted metabolomic studies with special focus on forensic applications
CN114720704B (en) Kit and method for measuring free testosterone in serum
CN112730710B (en) Detection method for rapid real-time quantification of target analytes in a sample by introducing a series of different isotopic labels
CN113049719A (en) Method and kit for detecting free testosterone
CN115343398A (en) Kit and method for simultaneously measuring multiple steroid hormones in serum
CN113720943A (en) Method for rapidly determining content of phthalate metabolites in urine
CN113341027A (en) Method and kit for detecting testosterone in saliva by high performance liquid chromatography tandem mass spectrometry
CN114689734A (en) Steroid substance source detection method and application thereof
CN113009036A (en) Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones
CN110068645B (en) Method for detecting cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry
CN112798695A (en) Method for simultaneously detecting benzo [ a ] pyrene and metabolite thereof and application thereof
KR101159064B1 (en) Quantification of active androgens in urine and blood by isotope dilution-mass spectrometry
Wen et al. Isolation and enrichment of Boldenone and its main metabolite in urinary samples to further determine the 13C/12C ratios by gas chromatography/combustion/isotope ratio mass spectrometry
CN113030343B (en) Liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in blood plasma
CN112345680B (en) Method for simultaneously detecting eight sterols in ganoderma lucidum
CN109324139A (en) Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf
CN112946109A (en) Detection method and application of endogenous glucocorticoid
CN110749666A (en) Liquid chromatography tandem mass spectrometry method for detecting busulfan in plasma
CN103808836B (en) Determination method of 3-alkylated adenine DNA adducts in urine
CN114563504B (en) Method and kit for determining content of free aldosterone in blood plasma
CN103926368A (en) Method for extracting biotin from corn steep liquor and thin layer chromatography (TLC) scanning detection method of biotin
CN113640415B (en) Method for detecting content of alpha-androstanol in truffle sporocarp

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20220701