CN112964815A - Method for detecting multiple steroid hormones in biological body fluid - Google Patents

Method for detecting multiple steroid hormones in biological body fluid Download PDF

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CN112964815A
CN112964815A CN201911285074.3A CN201911285074A CN112964815A CN 112964815 A CN112964815 A CN 112964815A CN 201911285074 A CN201911285074 A CN 201911285074A CN 112964815 A CN112964815 A CN 112964815A
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liquid
steroid hormones
detection
internal standard
biological fluid
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张晓哲
刘欣欣
刘丹
程孟春
赵楠
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N30/14—Preparation by elimination of some components
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • 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
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • 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
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood

Abstract

The invention discloses a method for detecting multiple steroid hormones in biological body fluid, belongs to the technical field of analytical chemistry and clinical examination, and can solve the problems of long detection time, low detection efficiency and low detection sensitivity of the conventional steroid hormone detection method. Preprocessing biological body fluid containing a substance to be detected, detecting and analyzing to obtain a detection result of multiple steroid hormones in the biological body fluid; the pretreatment includes liquid-liquid extraction. According to the invention, protein and other hydrophilic micromolecular impurities are removed by extraction, so that the interference of other impurities in the sample is reduced; a non-derivatization method is adopted, so that the operation steps are simplified, and the repeatability is improved; and the content of the nine steroid hormones can be measured simultaneously, and the detection result is accurate and has good repeatability and stability.

Description

Method for detecting multiple steroid hormones in biological body fluid
Technical Field
The invention relates to the technical field of analytical chemistry and clinical examination, in particular to a method for detecting multiple steroid hormones in biological body fluid.
Background
Steroid hormones are lipid-soluble small molecules generated by cholesterol through a series of enzyme catalysis, and have important functions of maintaining metabolism, regulating sexual function and the like. Steroid hormones are metabolized with each other, continuously produced or consumed, and thus maintained in a dynamic equilibrium state under the catalysis of various enzymes in the human body. There is therefore a clinical need to accurately measure the concentration of each steroid hormone and understand the direct metabolic relationships between its upstream and downstream products. This can provide help for the diagnosis of endocrine system diseases, male/female reproductive system diseases, tumors, fertility functions, and the like. However, since steroid hormones are related to a plurality of molecular species, have similar structures and low in vivo content, quantitative detection and analysis have certain difficulty.
At present, the steroid hormone is clinically detected by a multi-purpose immunoassay method, the method can be automatically operated, the detection is rapid, the cost is low, the defects are that the specificity is poor, the cross reaction is easy to occur, the structural analogues cannot be distinguished and detected, only one steroid hormone can be detected at one time, and more importantly, part of the hormones in the steroid hormone metabolic pathway have no specific immunoreaction, so the hormones cannot be detected by the immunoassay method.
Disclosure of Invention
In view of the above, the invention provides a method for detecting multiple steroid hormones in a biological fluid, which can solve the problems of long detection time, low detection efficiency and low detection sensitivity of the conventional method for detecting the steroid hormones.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for detecting multiple steroid hormones in biological fluids, comprising the steps of:
pretreating biological body fluid containing a substance to be detected, and detecting and analyzing to obtain a detection result of multiple steroid hormones in the biological body fluid;
the biological fluid contains steroid hormone;
the pretreatment comprises liquid-liquid extraction.
Protein and other hydrophilic micromolecular impurities are removed through liquid-liquid extraction, and the interference of other impurities in the biological body fluid is reduced.
As a still further scheme of the invention: the liquid-liquid extraction is at least one of salting-out assisted liquid-liquid extraction and tert-butyl methyl ether extraction;
the auxiliary salting-out reagent in the salting-out auxiliary liquid-liquid extraction comprises saturated high-valence phosphate solution and isopropanol;
wherein the volume ratio of the saturated high-valence phosphate solution to the isopropanol is 4: 1-1: 4.
Preferably, the volume ratio of the saturated high valent phosphate solution to the isopropanol is independently selected from: 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1: 4.
Specifically, the mixed solution of the saturated high-valent phosphate solution and isopropanol is referred to as a SALLE reagent.
As a still further scheme of the invention: the liquid-liquid extraction comprises:
adding a liquid-liquid extracting agent into the extract to be extracted, and whirling for 1-3 minutes;
preferably, the liquid-liquid extraction comprises:
and adding the liquid-liquid extracting agent into the extract to be extracted, carrying out vortex for 1-3 minutes, centrifuging, and carrying out vortex after redissolution.
Specifically, the liquid-liquid extraction comprises: and adding a liquid-liquid extracting agent into the extract to be tested, shaking and centrifuging to obtain supernatant, drying by using nitrogen, adding a complex solution, shaking and centrifuging to obtain supernatant, and thus obtaining the sample to be tested.
Preferably, the double solvent is 50% -100% acetonitrile solution.
As a still further scheme of the invention: the biological fluid comprises at least one of plasma, serum, urine, and saliva;
the steroid hormone is at least one selected from cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone.
As a still further scheme of the invention: the detection analysis comprises: quantitative analysis is carried out by adopting an isotope internal standard quantitative method.
As a still further scheme of the invention: the object to be detected comprises an isotope internal standard substance of steroid hormone;
the isotopic internal standard substance of steroid hormone is selected from steroid hormoneIs/are as follows13A C label or a deuterated label;
preferably, the isotopic internal standard of the steroid hormone is selected from the group consisting of d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, and,13C3-17-hydroxyprogesterone, d 3-androstenedione and d 9-progesterone.
Preferably, the isotopic internal standard of steroid hormone comprises d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, d,13At least one of C3-17-hydroxyprogesterone, d 3-androstenedione, and d 9-progesterone.
Specifically, the preparation method of the isotope internal standard working solution comprises the following steps: respectively preparing d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, and d-cortisone with methanol,131mg/mL of stock solution of C3-17-hydroxyprogesterone, d 3-androstenedione or d 9-progesterone, and diluting the stock solution into mixed solution of isotope internal standard by using methanol solution with volume fraction of 50-100%, wherein the concentration of the mixed solution is as follows: 1 ng/mL-100 ng/mL.
Preferably, the adding amount of the isotope internal standard solution is as follows: the volume ratio of the biological body fluid to the isotope internal standard fluid is 1-15: 1.
specifically, 20. mu.L of a biological fluid sample containing d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, and d,13Adding 1mL of SALLE reagent or 1mL of tert-butyl methyl ether into isotope internal standard working solution of C3-17-hydroxyprogesterone, d 3-androstenedione and d 9-progesterone, performing vortex for 1-3 minutes, centrifuging at 15000rpm for 5-10 minutes, taking supernatant, drying the supernatant at 40 ℃ with nitrogen, adding 30-100 muL of acetonitrile solution with the volume fraction of 50-100% for redissolution, performing vortex for 1 minute, centrifuging at 15000rpm for 5-10 minutes, and taking the supernatant to obtain a sample to be detected.
Specifically, the preparation method of the standard substance comprises the following steps: preparing 1mg/mL stock solutions of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone by using methanol respectively, and diluting the stock solutions into a series of standard solutions by using methanol solutions with volume fractions of 50-100% to obtain 5 concentration points.
Specifically, the extraction steps of the standard product are as follows: adding 20 mu L of series standard substance solution into 180 mu L of blank matrix, adding 20 mu L of isotope internal standard working solution, adding 1mL of SALLE reagent or 1mL of tert-butyl methyl ether, whirling for 1-3 minutes, centrifuging for 5-10 minutes at 15000rpm, taking supernatant, drying with nitrogen at 40 ℃, adding 30 mu L-100 mu L of acetonitrile solution with the volume fraction of 50-100% for redissolution, whirling for 1 minute, centrifuging for 5-10 minutes at 15000rpm, taking supernatant to obtain a sample to be detected, wherein the blank matrix is blank plasma, serum, urine or saliva without steroid hormones.
As a still further scheme of the invention: the quantitative analysis by adopting an isotope internal standard quantitative method comprises the following steps: and (4) taking the concentration of the standard substance as an x axis, and taking the peak area ratio of the standard substance to the internal standard substance as a y axis to draw a standard curve.
As a still further scheme of the invention: the detection analysis comprises:
detecting the pretreated sample to be detected by adopting a liquid chromatography tandem mass spectrometry method, and carrying out quantitative analysis by adopting an isotope internal standard quantitative method to obtain the steroid hormone content in the sample to be detected;
preferably, detection is by high performance liquid chromatography tandem mass spectrometry.
As a still further scheme of the invention: the liquid chromatography conditions were:
the chromatographic column is a reverse chromatographic column, and the mobile phase is composed of a mobile phase A and a mobile phase B according to a proportion and is subjected to gradient elution;
the mobile phase A is a volatile acid solution with the volume fraction of 0.1-2% or a volatile buffer salt solution with the volume fraction of 0.1-2 mmol/L;
the mobile phase B is methanol with 0.02 to 0.2 percent of formic acid by volume fraction;
the flow rate of the mobile phase is 0.3mL/min to 0.5mL/min, and the column temperature is 35 ℃ to 45 ℃;
the volatile acid solution is at least one of formic acid, acetic acid and trifluoroacetic acid;
the volatile buffer salt solution is at least one of ammonium formate, ammonium acetate and ammonium fluoride.
Preferably, the mobile phase a is independently selected from the group consisting of a 0.1% volatile acid solution, a 0.5% volatile acid solution, a 0.8% volatile acid solution, a 1% volatile acid solution, a 1.5% volatile acid solution, and a 2% volatile acid solution by volume fraction.
Preferably, mobile phase A is independently selected from 0.1mmol/L volatile buffered salt solution, 0.5mmol/L volatile buffered salt solution, 0.8mmol/L volatile buffered salt solution, 1mmol/L volatile buffered salt solution, 1.5mmol/L volatile buffered salt solution, 2mmol/L volatile buffered salt solution.
Preferably, mobile phase B is independently selected from methanol with a volume fraction of 0.02% formic acid, 0.05% formic acid, 0.08% formic acid, 0.1% formic acid, 0.15% formic acid, 0.2% formic acid.
Preferably, the flow rates of the mobile phases are independently selected from 0.3mL/min, 0.4mL/min, 0.5 mL/min.
Preferably, the column temperature is independently selected from 35 deg.C, 38 deg.C, 40 deg.C, 42 deg.C, 44 deg.C, and 45 deg.C.
Furthermore, the sample injection amount is 5-20 muL.
The elution gradient was:
0～1min：0％～65％B；
1～6.5min：65％～75％B；
6.5～10min：75％～100％B；
10～11min：100％B；
11～13min：65％B。
preferably, the reverse phase chromatography column is C18、C8And at least one of the phenyl columns, the particle size range is 1-5 mu m, the length range is 50-150 mm, and the diameter range is 2-5 mm.
As a still further scheme of the invention: the mass spectrum conditions are as follows: an electrospray ionization source, a positive ion mode and a multi-reaction monitoring scanning mode are adopted.
In positive ion mode, a quantitative ion pair comprising at least one of the following steroid hormones: cortisol ion pair, cortisone ion pair, 11-deoxycorticosterol ion pair, corticosterone ion pair, dehydroepiandrosterone ion pair, testosterone ion pair, 17-hydroxyprogesterone ion pair, androstenedione ion pair, and progesterone ion pair; and/or, a quantitative ion pair of a cortisol internal standard, a quantitative ion pair of a cortisone internal standard, a quantitative ion pair of an 11-deoxycorticosterol internal standard, a quantitative ion pair of a corticosterone internal standard, a quantitative ion pair of a dehydroepiandrosterone internal standard, a quantitative ion pair of a 17-hydroxyprogesterone internal standard, a quantitative ion pair of an androstenedione internal standard and a quantitative ion pair of a progesterone internal standard;
multiple reaction monitoring of target quantitation ions mass/charge ratio conditions of ion scanning MRMs include:
the mass/charge ratio of parent ions of the cortisol is 362.75-363.25, and the mass/charge ratio of corresponding daughter ions is 120.75-121.25;
the mass/charge ratio of parent ions of the cortisone is 360.75-361.25, and the mass/charge ratio of corresponding daughter ions is 162.85-163.35;
the mass/charge ratio of parent ions of the 11-deoxycorticosterol is 346.75-347.25, and the mass/charge ratio of corresponding daughter ions is 96.85-97.35;
the mass/charge ratio of the parent ion of the corticosterone is 347.05-347.55, and the mass/charge ratio of the corresponding daughter ion is 120.75-121.25;
the mass/charge ratio of the parent ion of the dehydroepiandrosterone is 288.85-289.35, and the mass/charge ratio of the corresponding daughter ion is 212.95-213.45;
the mass/charge ratio of parent ions of testosterone is 288.75-289.25, and the mass/charge ratio of corresponding daughter ions is 96.85-97.35;
the mass/charge ratio of the parent ion of the 17-hydroxyprogesterone is 330.85-331.35, and the mass/charge ratio of the corresponding daughter ion is 96.85-91.35;
the mass/charge ratio of parent ions of the androstenedione is 286.95-287.45, and the mass/charge ratio of corresponding daughter ions is 96.75-97.25;
the mass/charge ratio of the parent ion of the progesterone is 314.95-315.45, and the mass/charge ratio of the corresponding daughter ion is 96.75-97.25;
the mass/charge ratio of parent ions of the d 4-cortisol is 366.85-367.35, and the mass/charge ratio of corresponding daughter ions is 120.85-121.35;
the mass/charge ratio of parent ions of d 8-cortisone is 368.75-369.25, and the mass/charge ratio of corresponding daughter ions is 168.95-169.45;
the mass/charge ratio of parent ions of the d 5-11-deoxycorticosterol is 351.85-352.35, and the mass/charge ratio of corresponding daughter ions is 99.85-100.35;
the mass/charge ratio of parent ions of the d 8-corticosterone is 354.85-355.35, and the mass/charge ratio of corresponding daughter ions is 124.75-125.25;
the mass/charge ratio of parent ions of the d 6-dehydroepiandrosterone is 294.85-295.35, and the mass/charge ratio of corresponding daughter ions is 218.95-219.45;
the mass/charge ratio of parent ions of the d 3-testosterone is 288.75-289.25, and the mass/charge ratio of corresponding daughter ions is 99.85-100.35;
13the mass/charge ratio of parent ions of C3-17-hydroxyprogesterone is 333.85-334.35, and the mass/charge ratio of corresponding daughter ions is 99.85-100.35;
the mass/charge ratio of parent ions of the d 3-androstenedione is 289.95-290.45, and the mass/charge ratio of corresponding daughter ions is 99.85-100.35;
the mass/charge ratio of parent ions of the d 9-progesterone is 324.15-324.65, and the mass/charge ratio of corresponding daughter ions is 99.85-100.35.
Further, the air curtain pressure (CUR) is 20 kPa-30 kPa; collision gas (CAD) is Medium; the spraying voltage (IS) IS 5000V-6000V; the heating gas temperature is 500-550 ℃.
Preferably, the mass spectrometric conditions of the LC-MS detection further comprise at least one of the following steroid hormone quantification ion pair declustering voltages and collision energies:
the declustering voltage of the cortisol quantitative ion pair is 90V-130V, and the collision energy is 28V-38V;
the cluster removing voltage of the cortisone quantitative ion pair is 141V-181V, and the collision energy is 28V-38V;
the cluster removing voltage of the 11-deoxycorticosterol quantitative ion pair is 105V-145V, and the collision energy is 23V-33V;
the cluster removing voltage of the corticosterone quantitative ion pair is 101V-141V, and the collision energy is 26V-36V;
the cluster removing voltage of the dehydroepiandrosterone quantitative ion pair is 88V-128V, and the collision energy is 21V-31V;
the cluster removing voltage of the testosterone quantitative ion pair is 120V-160V, and the collision energy is 22V-32V;
the cluster removing voltage of the 17-hydroxyprogesterone quantitative ion pair is 116V-156V, and the collision energy is 25V-35V;
the cluster removing voltage of the androstenedione quantitative ion pair is 129V-169V, and the collision energy is 15V-25V;
the cluster removing voltage of the progesterone quantitative ion pair is 125V-165V, and the collision energy is 23V-33V;
the declustering voltage of the d 4-cortisol quantitative ion pair is 125-165V, and the collision energy is 25-35V;
the cluster removing voltage of the d 8-cortisone quantitative ion pair is 134V-174V, and the collision energy is 28V-38V;
the declustering voltage of the d 5-11-deoxycorticosterol quantitative ion pair is 120V-160V, and the collision energy is 23V-33V;
the declustering voltage of the d 8-corticosterone quantitative ion pair is 103V-143V, and the collision energy is 26V-36V;
the declustering voltage of the d 6-dehydroepiandrosterone quantitative ion pair is 80V-120V, and the collision energy is 19V-29V;
the declustering voltage of the d 3-testosterone quantitative ion pair is 120V-160V, and the collision energy is 22V-32V;
13the declustering voltage of the C3-17-hydroxyprogesterone quantitative ion pair is 130V-170V, and the collision energy is 23V-33V;
the declustering voltage of the d 3-androstenedione quantitative ion pair is 100V-140V, and the collision energy is 22V-32V;
the declustering voltage of the d 9-progesterone quantitative ion pair is 120V-160V, and the collision energy is 23V-33V.
Further, the linear range of the detection method of the invention is as follows: 0.1-500 ng/mL of cortisol, 0.1-100 ng/mL of cortisone, 0.01-10 ng/mL of 11-deoxycorticosterol, 0.1-100 ng/mL of corticosterone, 0.5-50 ng/mL of dehydroepiandrosterone, 0.02-20 ng/mL of testosterone, 0.02-10 ng/mL of 17-hydroxyprogesterone, 0.05-10 ng/mL of androstenedione and 0.05-20 ng/mL of progesterone.
The beneficial effects of the invention include but are not limited to:
(1) the method for detecting the multiple steroid hormones in the biological body fluid, provided by the invention, comprises the steps of pretreating, detecting and analyzing the biological body fluid containing an object to be detected to obtain a detection result of the multiple steroid hormones in the biological body fluid; the pretreatment includes liquid-liquid extraction. According to the invention, protein and other hydrophilic micromolecular impurities are removed by extraction, so that the interference of other impurities in the sample is reduced; a non-derivatization method is adopted, so that the operation steps are simplified, and the repeatability is improved; and the content of the nine steroid hormones can be measured simultaneously, and the detection result is accurate and has good repeatability and stability.
(2) The method is suitable for synchronous batch detection of steroid hormones, has excellent separation capacity on isomers of the steroid hormones, is high in resolution and sensitivity, and can avoid the phenomenon that the detection result is higher due to the interference of the isomers.
(3) The method can meet the requirement of detection limit without derivatization, has simplified steps compared with other liquid chromatography-mass spectrometry detection methods requiring derivatization, shortens the pretreatment time, and can improve the repeatability of detection results. Compared with the traditional immunoassay and other methods for detecting single steroid hormone, the method has the advantages of reducing the experiment cost, shortening the analysis time, improving the detection flux and meeting the detection requirement of clinical steroid hormone.
Drawings
FIG. 1 is an ion flow diagram of the extraction of isomeric corticosterone and 11-deoxycorticol;
figure 2 is an ion flow diagram of the extraction of the isomers dehydroepiandrosterone and testosterone.
Detailed Description
The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
Example 1:
this example demonstrates the accuracy of 9 steroid hormones (cortisol, cortisone, 11-deoxycorticol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione, and progesterone) using the method of the invention.
(1) The preparation method of the isotope internal standard working solution comprises the following steps: respectively preparing d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, and d-cortisone with methanol,13C3-17-hydroxyprogesterone, d 3-androstenedione and d 9-progesterone, wherein the stock solutions are 1mg/mL, and are diluted into mixed solutions of isotope internal standards by using methanol solutions with volume fractions of 50%, and the concentrations of the mixed solutions are 1000ng/mL, 200ng/mL, 10ng/mL, 100ng/mL, 20ng/mL, 25ng/mL, 10ng/mL and 10ng/mL respectively.
(2) Standard solution: preparing 1mg/mL stock solutions of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone by using methanol respectively, diluting the stock solutions into a series of standard solutions by using a methanol solution with a volume fraction of 50%, wherein the volume fraction of the methanol solution is 5 concentration points, and the concentration of each steroid hormone is as follows: cortisol 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 2500 ng/mL; cortisone 2ng/mL, 4ng/mL, 20ng/mL, 100ng/mL, 500 ng/mL; 11-deoxycorticosterol 0.1ng/mL, 0.2ng/mL, 1ng/mL, 5ng/mL, 25 ng/mL; corticosterone 1ng/mL, 2ng/mL, 10ng/mL, 50ng/mL, 250 ng/mL; dehydroepiandrosterone 1ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250 ng/mL; testosterone is 0.5ng/mL, 1ng/mL, 5ng/mL, 25ng/mL, 125 ng/mL; 17-hydroxyprogesterone 0.2ng/mL, 0.4ng/mL, 2ng/mL, 10ng/mL, 50 ng/mL; androstenedione 0.5ng/mL, 1ng/mL, 2ng/mL, 10ng/mL, 50 ng/mL; progesterone 0.5ng/mL, 1ng/mL, 5ng/mL, 25ng/mL, 125 ng/mL.
(3) Quality Control (QC) solution: preparing 1mg/mL stock solutions of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone by using methanol respectively, diluting the stock solutions into quality control solution by using a methanol solution with a volume fraction of 50%, wherein the volume fraction of the methanol solution is 3 concentration points, and the concentration of each steroid hormone is as follows: cortisol 50ng/mL, 250ng/mL, 1250 ng/mL; cortisone 10ng/mL, 50ng/mL, 250 ng/mL; 11-deoxycorticosterol 0.5ng/mL, 2.5ng/mL, 12.5 ng/mL; corticosterone 5ng/mL, 25ng/mL, 125 ng/mL; dehydroepiandrosterone 2ng/mL, 10ng/mL, 125 ng/mL; testosterone is 2.5ng/mL, 12.5ng/mL, 62.5 ng/mL; 17-hydroxyprogesterone 1ng/mL, 5ng/mL, 25 ng/mL; androstenedione 1ng/mL, androstenedione 5ng/mL, androstenedione 25 ng/mL; progesterone 2.5ng/mL, 12.5ng/mL, 62.5 ng/mL.
(4) Pretreatment of a standard substance and a quality control substance: adding 20 mu L of standard substance solution or quality control substance solution into 180 mu L of blank plasma, adding 20 mu L of isotope internal standard working solution, adding 0.5mL of saturated potassium phosphate solution and 0.5mL of isopropanol, carrying out vortex for 3 minutes, centrifuging at 15000rpm for 10 minutes, taking supernatant, carrying out nitrogen blow-drying at 40 ℃, adding 50 mu L of 90% acetonitrile water solution for redissolution, carrying out vortex for 1 minute, centrifuging at 15000rpm for 10 minutes, and taking supernatant to obtain a sample to be detected.
(5) High performance liquid chromatography tandem mass spectrometry
The apparatus used was ABI Q TRAP 5500(AB SCIEX);
high performance liquid chromatography conditions: the mobile phase A is a formic acid solution with the volume fraction of 0.2 percent; the mobile phase B is methanol with the volume fraction of 0.2 percent formic acid; the chromatographic column is as follows: InfinityLabPoroshell 120EC-C18,50 mm. times.2.1 mm, 2.7 μm; the flow rate is 0.3mL/min, the column temperature is 35 ℃, and the sample injection amount is 5 mu L; the elution gradient is 0-1 min: 0% -65% of B; 1-6.5 min: 65% -75% of B; 6.5-10 min: 75% -100% of B; 10-11 min: 100% B.
The mass spectrum conditions are as follows: electrospray ionization source, positive ion mode, scanning mode using Multiple Reaction Monitoring (MRM); air curtain gas (CUR) is 20 kPa; collision gas (CAD) is Medium; the spray voltage (IS) IS 5000V; the heating gas temperature is 500 ℃; the quantitative ion pair, declustering voltage (DP) and Collision Energy (CE) parameters for the 9 steroid hormones and their isotopic internal standards are shown in table 1.
TABLE 19 MRM analysis parameters of steroid hormones and their isotopic internal standards
Figure BDA0002317757610000101
The steroid hormone contains a plurality of pairs of isomers, the same daughter ions and parent ions can be generated in a mass spectrum Multiple Reaction Monitoring (MRM) scanning mode, if the separation degree of a chromatographic method is not enough, mutual interference is easy to occur, and the detection result is higher. There are two pairs of isomers in the present invention: 11-deoxycorticosterol and corticosterone, dehydroepiandrosterone and testosterone, shown in figures 1 and 2, are ion flow diagrams extracted from them, and it can be seen that both pairs of isomers achieve baseline separation.
Taking the concentration of the standard substance as an x axis, taking the peak area ratio of the standard substance and the internal standard substance as a y axis to draw a standard curve, and calculating the content of each hormone; retention time, standard curve and correlation for 9 steroid hormones as shown in table 2, cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone, respectively, showed good linearity in the following ranges: 1 ng/m-250 ng/mL, 0.2 ng/m-50 ng/mL, 0.01 ng/m-2.5 ng/mL, 0.1 ng/m-25 ng/mL, 0.5 ng/m-25 ng/mL, 0.05 ng/m-12.5 ng/mL, 0.02 ng/m-5 ng/mL, 0.1 ng/m-5 ng/mL, 0.05 ng/m-12.5 ng/mL.
The recovery rate of 9 steroid hormone substrate standard curves is shown in tables 3-11, and the results show that each steroid hormone standard curve presents good linearity in a linear range, and the standard recovery rate of each point of the standard curve is within a range of 85-115%, which indicates that the method is reliable; the quality control results are shown in table 12, the recovery rate is calculated by substituting the quality control detection data into the standard curve, and as can be seen from table 12, the recovery rate is in the range of 85% -115%, which indicates that the detection system is stable and the results are reliable.
TABLE 29 Standard Curve for steroid hormones
Figure BDA0002317757610000111
TABLE 3 recovery (%) of cortisol on standard substrate for standard curve
Figure BDA0002317757610000112
Figure BDA0002317757610000121
TABLE 4 recovery (%) of cortisone on standard substrate for standard curve
Figure BDA0002317757610000122
TABLE 511-recovery (% of deoxycorticol in standard substrate plus standard)
Figure BDA0002317757610000123
TABLE 6 recovery (%) of corticosterone in standard medium and standard plus standard
Figure BDA0002317757610000124
TABLE 7 substrate standard plus standard recovery (%)
Figure BDA0002317757610000125
TABLE 8 recovery (%) of testosterone on a medium standard curve plus standard
Figure BDA0002317757610000131
TABLE 917 substrate standard plus standard recovery (%) -of hydroxyprogesterone
Figure BDA0002317757610000132
Recovery (%) -for epi10 androstenedione on a substrate standard plus standard basis
Figure BDA0002317757610000133
TABLE 11 recovery (%) of progesterone in standard medium and standard curve
Figure BDA0002317757610000134
TABLE 129 Quality Control (QC) results for steroid hormones
Figure BDA0002317757610000141
Example 2:
in this example, 13 plasma samples were tested for 9 steroid hormone content.
The biological fluid sample is collected in the seventh national hospital of Dalian city, and all volunteers take blood on an empty stomach at 7: 00-8: 00 am. Collecting whole blood with anticoagulant-containing vacuum blood collection tube, centrifuging at 3000rpm for ten minutes, collecting upper layer plasma, subpackaging, and storing at-80 deg.C for use.
The preparation method of the isotope internal standard working solution, the preparation method of the standard solution, the pretreatment method of the standard and the parameter setting of the high performance liquid chromatography tandem mass spectrometry are the same as those in example 1.
Pretreatment of plasma samples: adding 20 mu L of isotope internal standard working solution into 200 mu L of plasma sample, adding 0.5mL of saturated potassium phosphate solution and 0.5mL of isopropanol, carrying out vortex centrifugation for 3 minutes at 15000rpm for 10 minutes, taking supernatant, carrying out nitrogen blow-drying at 40 ℃, adding 50 mu L of 90% acetonitrile solution for redissolution, carrying out vortex centrifugation for 1 minute at 15000rpm for 10 minutes, and taking supernatant to obtain a sample to be detected.
The 9 steroid hormone contents of the 13 plasma samples are shown in Table 13.
TABLE 1313 plasma samples 9 steroid hormone content
Figure BDA0002317757610000151
Example 3:
in this example, 13 serum samples were tested for the content of 9 steroid hormones.
Samples were collected in central hospitals in Dalian cities, and all volunteers sampled blood on an empty stomach at 7: 00-8: 00 am. Collecting whole blood with a vacuum blood collection tube without anticoagulant, standing for coagulation, centrifuging at 3000rpm for ten minutes, taking upper layer serum, subpackaging, and storing at-80 ℃ for later use.
The preparation method of the isotope internal standard working solution, the preparation method of the standard solution, the pretreatment method of the standard and the parameter setting of the high performance liquid chromatography tandem mass spectrometry are the same as those in example 1.
Pretreatment of a standard substance and a quality control substance: adding 20 mu L of standard substance solution or quality control substance solution into 180 mu L of blank plasma, adding 20 mu L of isotope internal standard working solution, adding 1mL of tert-butyl methyl ether, whirling for 3 minutes, centrifuging at 15000rpm for 10 minutes, taking supernatant, drying with nitrogen at 40 ℃, adding 50 mu L of 90% acetonitrile water solution for redissolution, whirling for 1 minute, centrifuging at 15000rpm for 10 minutes, and taking supernatant to obtain a sample to be detected.
Pretreatment of serum samples: adding 20 mul of isotope internal standard working solution into 200 mul of serum sample, adding 0.5mL of saturated potassium phosphate solution and 0.5mL of isopropanol, whirling for 3 minutes, centrifuging at 15000rpm for 10 minutes, taking supernatant, drying with nitrogen at 40 ℃, adding 90% acetonitrile solution for 50 mul of redissolution, whirling for 1 minute, centrifuging at 15000rpm for 10 minutes, and taking supernatant to obtain a sample to be detected.
The levels of 9 steroid hormones in 13 serum samples are shown in Table 14.
TABLE 1413 serum samples 9 steroid hormone levels
Figure BDA0002317757610000161
The above description is only for the purpose of illustrating the present invention and is not intended to limit the present invention in any way, and the present invention is not limited to the above description, but rather should be construed as being limited to the scope of the present invention.

Claims (10)

1. A method for detecting multiple steroid hormones in a biological fluid, comprising the steps of:
pretreating biological body fluid containing a substance to be detected, and detecting and analyzing to obtain a detection result of multiple steroid hormones in the biological body fluid;
the biological fluid contains steroid hormone;
the pretreatment comprises liquid-liquid extraction.
2. The method for detecting multiple steroid hormones as claimed in claim 1, wherein said liquid-liquid extraction is at least one selected from the group consisting of salting out assisted liquid-liquid extraction and tert-butyl methyl ether extraction;
the auxiliary salting-out reagent in the salting-out auxiliary liquid-liquid extraction comprises saturated high-valence phosphate solution and isopropanol;
wherein the volume ratio of the saturated high-valence phosphate solution to the isopropanol is 4: 1-1: 4.
3. The method for detecting multiple steroid hormones in a biological fluid as claimed in claim 1 or 2, wherein said liquid-liquid extraction comprises:
adding a liquid-liquid extracting agent into the extract to be extracted, and whirling for 1-3 minutes;
preferably, the liquid-liquid extraction comprises:
and adding the liquid-liquid extracting agent into the extract to be extracted, carrying out vortex for 1-3 minutes, centrifuging, and carrying out vortex after redissolution.
4. The method for detecting multiple steroid hormones as claimed in claim 1, wherein said biological fluid includes at least one of plasma, serum, urine and saliva;
the steroid hormone is at least one selected from cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone.
5. The method for the detection of multiple steroid hormones in a biological fluid as set forth in claim 1, wherein the detection analysis comprises: quantitative analysis is carried out by adopting an isotope internal standard quantitative method.
6. The method for detecting multiple steroid hormones in a biological fluid as claimed in claim 5, wherein an isotopic internal standard substance of a steroid hormone is included in said substance to be detected;
the isotopic internal standard of the steroid hormone is selected from the group consisting of steroid hormones13A C label or a deuterated label;
preferably, the isotopic internal standard of steroid hormone comprises d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone, d,13At least one of C3-17-hydroxyprogesterone, d 3-androstenedione, and d 9-progesterone.
7. The method for detecting multiple steroid hormones in a biological fluid according to claim 5, wherein the quantitative analysis by an isotopic internal standard method comprises: and (4) taking the concentration of the standard substance as an x axis, and taking the peak area ratio of the standard substance to the internal standard substance as a y axis to draw a standard curve.
8. The method for the detection of multiple steroid hormones in a biological fluid as set forth in claim 1, wherein the detection analysis comprises:
detecting the pretreated sample to be detected by adopting a liquid chromatography tandem mass spectrometry method, and carrying out quantitative analysis by adopting an isotope internal standard quantitative method to obtain the steroid hormone content in the sample to be detected;
preferably, detection is by high performance liquid chromatography tandem mass spectrometry.
9. The method for detecting multiple steroid hormones in a biological fluid as claimed in claim 8, wherein the liquid chromatography conditions are as follows:
the chromatographic column is a reverse chromatographic column, and the mobile phase is composed of a mobile phase A and a mobile phase B according to a proportion and is subjected to gradient elution;
the mobile phase A is a volatile acid solution with the volume fraction of 0.1-2% or a volatile buffer salt solution with the volume fraction of 0.1-2 mmol/L;
the mobile phase B is methanol with 0.02 to 0.2 percent of formic acid by volume fraction;
the flow rate of the mobile phase is 0.3mL/min to 0.5mL/min, and the column temperature is 35 ℃ to 45 ℃;
the volatile acid solution is at least one of formic acid, acetic acid and trifluoroacetic acid;
the volatile buffer salt solution is at least one of ammonium formate, ammonium acetate and ammonium fluoride.
10. The method for the detection of multiple steroid hormones in a biological fluid as claimed in claim 8, wherein said mass spectrometric conditions are: an electrospray ionization source, a positive ion mode and a multi-reaction monitoring scanning mode are adopted.
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