CN112964809A - Kit for detecting multiple steroid hormones in biological body fluid and use method thereof - Google Patents
Kit for detecting multiple steroid hormones in biological body fluid and use method thereof Download PDFInfo
- Publication number
- CN112964809A CN112964809A CN201911285102.1A CN201911285102A CN112964809A CN 112964809 A CN112964809 A CN 112964809A CN 201911285102 A CN201911285102 A CN 201911285102A CN 112964809 A CN112964809 A CN 112964809A
- Authority
- CN
- China
- Prior art keywords
- liquid
- solution
- working solution
- internal standard
- standard
- 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
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
- 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
- G01N30/88—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
- 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
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)
- Steroid Compounds (AREA)
Abstract
The invention discloses a kit for detecting multiple steroid hormones in biological body fluid and a use method thereof, 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. The kit for detecting multiple steroid hormones in biological body fluid comprises standard substance/standard substance working solution, quality control substance/quality control substance working solution, internal standard substance/internal standard substance working solution, matrix, liquid-liquid extraction liquid and eluent. The invention utilizes the liquid chromatogram tandem mass spectrum technology, directly detects the steroid hormone in the organism liquid sample according to the inherent physicochemical property/charge ratio of the analyte, adopts a non-derivatization method, has simple and convenient operation, can simultaneously detect the content of multiple steroid hormones in the organism liquid sample, greatly shortens the detection time and can meet the clinical detection requirement on the steroid hormone.
Description
Technical Field
The invention relates to the technical field of analytical chemistry and clinical examination, in particular to a kit for detecting multiple steroid hormones in biological fluid and a using method thereof.
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 clinical multi-purpose immunoassay method for detecting steroid hormones can be operated automatically, the detection is rapid, the cost is low, the defects are that the specificity is poor, cross reaction is easy to occur, structural analogues cannot be distinguished, only one steroid hormone can be detected at a time, and the detection time is long for detecting the multi-steroid hormone, so the requirements of clinical detection efficiency and detection accuracy cannot be met, and more importantly, part of hormones in a 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 kit for detecting multiple steroid hormones in biological body fluid and a use method thereof, which can solve the problems of long detection time, low detection efficiency and low detection sensitivity of the existing steroid hormone detection method.
In order to achieve the purpose, the invention provides the following technical scheme:
a kit for detecting multiple steroid hormones in biological body fluid comprises a liquid-liquid extraction agent.
As a still further scheme of the invention: the liquid-liquid extracting agent is at least one of salting-out auxiliary liquid-liquid extracting agent and tert-butyl methyl ether extracting agent;
preferably, the liquid-liquid extracting agent is selected from saturated high-valent phosphate solution and at least one of isopropanol and tert-butyl methyl ether;
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 salting-out auxiliary liquid-liquid extraction agent is also called SALLE reagent.
As a still further scheme of the invention: 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 kit for detecting multiple steroid hormones in biological body fluid also comprises: at least one of standard substance/standard substance working solution, quality control substance/quality control substance working solution, internal standard substance/internal standard substance working solution, matrix, eluent and double solvent.
As a still further scheme of the invention: the standard substance working solution is a standard substance methanol solution with the volume fraction of 50-100%;
the quality control product working solution is a quality control product methanol solution with the volume fraction of 50-100%;
the internal standard substance working solution is an internal standard substance methanol solution with the volume fraction of 50-100%;
the blank matrix comprises: blank plasma, serum, urine or saliva depleted of steroid hormones;
the eluent comprises: eluent 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, and eluent B is methanol containing formic acid with the volume fraction of 0.02-0.2%;
furthermore, the eluent A and the eluent B are respectively 500-1000 mL.
Preferably, the eluent 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, eluent A is independently selected from 0.1mmol/L volatile buffer salt solution, 0.5mmol/L volatile buffer salt solution, 0.8mmol/L volatile buffer salt solution, 1mmol/L volatile buffer salt solution, 1.5mmol/L volatile buffer salt solution, 2mmol/L volatile buffer salt solution.
Preferably, eluent 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.
The double solvent comprises an aqueous solution of acetonitrile;
specifically, the double solvent is acetonitrile solution with the concentration of 50-100%.
Preferably, the double solvent is 90% acetonitrile solution.
Preferably, 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 internal standard is an isotopic internal standard for a steroid hormone;
the isotopic internal standard of steroid hormone comprises the isotope of steroid hormone13At least one of a C label and a deuterated label;
preferably, the internal standard comprises d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone,13at least one of C3-17-hydroxyprogesterone, d 3-androstenedione, and d 9-progesterone.
As a still further scheme of the invention: the quality control product working solution is a methanol solution containing multiple steroid hormones corresponding to the standard product working solution, and the concentration of the methanol solution is at least 1;
preferably, the standard working solution comprises at least 4 steroid hormone standard solutions of different concentrations; the quality control product working solution comprises at least 2 steroid hormone quality control product solutions with different concentrations.
As a still further scheme of the invention: the kit for detecting multiple steroid hormones in biological body fluid comprises:
1) standard working solution: methanol solution containing cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone with volume fraction of 50-100%, with concentration of 4 or 5;
2) quality control product working solution: methanol solution containing cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone with volume fraction of 50-100%, with concentration of 2 or 3;
3) internal standard substance working solution: methanol solution containing 50-100% of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone isotope internal standard by volume fraction;
4) blank matrix: blank plasma, serum, urine or saliva depleted of steroid hormones;
5) liquid-liquid extracting agent: salting out at least one of an auxiliary liquid-liquid extraction reagent or tert-butyl methyl ether;
6) eluent: the eluent 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, and the eluent B is a methanol solution containing formic acid with the volume fraction of 0.02-0.2%.
The invention also discloses a detection method of the multiple steroid hormones in the biological body fluid, which adopts the kit for detecting the multiple steroid hormones in the biological body fluid to carry out detection, and the detection method comprises the following steps:
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.
As a still further scheme of the invention: the detection method comprises the following steps:
preferably, the pretreatment of the standard substance or quality control substance working solution comprises the following steps: extracting the mixed liquid of the standard substance or the quality control substance working solution and the corresponding isotope internal standard solution by using a liquid-liquid extraction agent, shaking and centrifuging to obtain supernatant, drying by using nitrogen, adding a complex solution, shaking and centrifuging to obtain the supernatant, and thus obtaining the standard substance to be detected.
Specifically, 20 microliter of series standard substance working solution or quality control substance working solution is added into 180 microliter of blank substrate, 20 microliter of internal standard substance working solution is added, 1mL of SALLE reagent or 1mL of tert-butyl methyl ether liquid extraction agent is added, the mixture is swirled for 1-3 minutes and is centrifuged at 15000rpm for 5-10 minutes, then supernatant is taken and dried by nitrogen at 40 ℃, acetonitrile solution with the volume fraction of 50% -100% is added for redissolution, and the supernatant is taken after the mixture is swirled for 1 minute and is centrifuged at 15000rpm for 5-10 minutes to obtain the standard substance to be detected.
As a still further scheme of the invention: 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.
preferably, the volume ratio of the biological fluid to the isotope internal standard solution is independently selected from 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15: 1.
Step 2, pretreatment of the biological fluid sample: performing liquid-liquid extraction, centrifugation and redissolution on a mixed liquid of the biological fluid sample and a corresponding isotope internal standard liquid to obtain a sample to be detected;
preferably, the biological fluid sample pretreatment: extracting a mixed liquid of the biological fluid sample and the corresponding isotope internal standard liquid by using a liquid-liquid extracting agent, oscillating and centrifuging to obtain a supernatant, drying by using nitrogen, adding a complex solution, oscillating and centrifuging to obtain the supernatant, and thus obtaining a sample to be detected.
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 liquid-liquid extraction agent into internal standard substance working solution of C3-17-hydroxyprogesterone, d 3-androstenedione and d 9-progesterone, whirling for 1-3 minutes, centrifuging at 15000rpm for 5-10 minutes, and taking supernatantBlowing the mixture with nitrogen at 40 ℃, adding 30-100 mu L 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 supernatant to obtain a sample to be detected.
Step 3, detecting the sample to be detected by adopting a high performance liquid chromatography-tandem mass spectrometry method, and quantifying by adopting an isotope internal standard quantification method;
preferably, the liquid chromatography conditions set: the chromatographic column is a reversed phase chromatographic column, the column temperature is 35-45 ℃, the flow rate of the eluent is 0.3-0.5 mL/min, and the elution gradient is set as follows:
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 chromatographic 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.
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.
Preferably, the flow rate of the eluent is independently selected from 0.3mL/min, 0.4mL/min, 0.5 mL/min.
Furthermore, the sample injection amount is 5-20 muL.
Preferably, the mass spectrometry conditions set: an electrospray ionization source, a positive ion mode and a multi-reaction monitoring scanning mode are adopted.
Specifically, 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 ℃.
Further, in positive ion mode, quantitative ion pairs of the various steroid hormones corresponding to the standard working solution are included: 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 mass spectrometry conditions also include the declustering voltage and collision energy of the multi-steroid hormone quantitative ion pairs corresponding to the standard working solution:
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 kit for detecting multiple steroid hormones in biological body fluid comprises standard substance/standard substance working solution, quality control substance/quality control substance working solution, internal standard substance/internal standard substance working solution, matrix, liquid-liquid extraction agent and eluent. The invention utilizes the liquid chromatography-tandem mass spectrometry technology, adopts a non-derivatization method, has simple and convenient operation, can simultaneously detect the content of multiple steroid hormones in an organism liquid sample, reduces the experiment cost, shortens the analysis time, improves the detection flux, has high detection result accuracy, good repeatability and good stability, and can meet the clinical detection requirement on the steroid hormones.
(2) The method has excellent separation capability on the isomers of the steroid hormone, the resolution and the sensitivity of the detection method are high, and the phenomenon that the measurement result is higher due to the interference of the isomers can be avoided.
(3) The detection method used by the kit is a liquid chromatography-tandem mass spectrometry method, and the steroid hormone in the biological fluid sample is directly detected according to the inherent physicochemical property/charge ratio of the analyte, so that the problems of low sensitivity, poor specificity and low accuracy caused by the traditional indirect detection methods such as the existing immunoassay method and the like can be solved.
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-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione, and progesterone) using the kit and method of use of the invention.
1. Kit and composition thereof
(1) Standard working solution: 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 standard working solutions by using 50% methanol solutions, wherein 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.
(2) Quality control product working solution: preparing 1mg/mL stock solutions of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone by using methanol respectively, and then diluting the stock solutions into a quality control product working solution by using a 50% methanol solution, wherein 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.
(3) Internal standard substance working solution: contains d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone,13The concentrations of methanol aqueous solutions of C3-17-hydroxyprogesterone, d 3-androstenedione and d 9-progesterone are 1000ng/mL, 200ng/mL, 10ng/mL, 100ng/mL, 20ng/mL, 25ng/mL, 10ng/mL and 10ng/mL respectively.
(4) Blank matrix: blank plasma to remove steroid hormones.
(5) Liquid-liquid extracting agent: the volume ratio of saturated high-valence phosphate solution to isopropanol is 1:1.
(6) Eluent: eluent A is 0.2% formic acid solution, and eluent B is methanol with 0.2% formic acid volume fraction.
(7) And (3) double solvent: 90% aqueous acetonitrile.
2. The use method of the kit comprises the following steps:
1) pretreating a standard substance and a quality control substance working solution: adding 20 mu L of standard substance or quality control substance working solution into 180 mu L of blank plasma, adding 20 mu L of internal standard substance working solution, adding 1mL of SALLE reagent, 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 the standard substance to be detected.
2) Setting liquid chromatography conditions:
the instrument used was ABIQTRAP 5500(AB SCIEX);
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% of B; 11-13 min: 65% of B.
3) Setting mass spectrum conditions: 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
4) And (3) computer detection: and (3) the pretreated standard substance to be detected enters a high performance liquid chromatography-tandem mass spectrometer for detection and analysis, and the peak areas of various steroid hormones and the peak area of the internal standard substance in the chromatogram and the detection standard substance are recorded.
5) And (4) analyzing results:
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, whose extracted ion flow diagrams are shown in fig. 1 and 2, it can be seen that both pairs of isomers achieve baseline separation.
The concentration of 5 standard working solutions is taken as an x axis, the concentration of the standard is taken as the x axis, the peak area ratio of the standard to an internal standard substance is taken as a y axis to draw a standard curve, the retention time, the standard curve and the correlation of 9 steroid hormones are shown in table 2, and cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone respectively show 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
TABLE 3 recovery (%) of cortisol on standard substrate for standard curve
TABLE 4 recovery (%) of cortisone on standard substrate for standard curve
TABLE 511-recovery (% of deoxycorticol in standard substrate plus standard)
TABLE 6 recovery (%) of corticosterone in standard medium and standard plus standard
TABLE 7 substrate standard plus standard recovery (%)
TABLE 8 recovery (%) of testosterone on a medium standard curve plus standard
TABLE 917 substrate standard plus standard recovery (%) -of hydroxyprogesterone
Recovery (%) -for epi10 androstenedione on a substrate standard plus standard basis
TABLE 11 recovery (%) of progesterone in standard medium and standard curve
TABLE 129 Quality Control (QC) results for steroid hormones
Example 2:
in this example, the kit and the method of using the same are the same as those in example 1, except that the method of pretreating a biological fluid sample is included in this example.
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 pretreatment method of the biological fluid sample comprises the following steps: adding 20 mu L of internal standard substance working solution into 200 mu L of plasma sample, adding 1mL of liquid-liquid extracting agent, 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.
The 9 steroid hormone contents of the 13 plasma samples are shown in Table 12.
TABLE 1313 plasma samples 9 steroid hormone content
Example 3:
in this example, the kit and the method of using the same as in example 2 are different from those in the biological fluid sample, which is serum.
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 levels of 9 steroid hormones in 13 serum samples are shown in Table 13.
TABLE 1413 serum samples 9 steroid hormone levels
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 kit for detecting multiple steroid hormones in biological body fluid is characterized by comprising a liquid-liquid extraction agent.
2. The kit for detecting multiple steroid hormones in a biological fluid according to claim 1, wherein said liquid-liquid extracting agent is at least one selected from the group consisting of salting-out-assisting liquid-liquid extracting agent and t-butyl methyl ether extracting agent;
preferably, the liquid-liquid extracting agent is selected from saturated high-valent phosphate solution and at least one of isopropanol and tert-butyl methyl ether;
wherein the volume ratio of the saturated high-valence phosphate solution to the isopropanol is 4: 1-1: 4.
3. The kit for detecting multiple steroid hormones in biological fluids as claimed in claim 1, wherein the steroid hormone is at least one selected from the group consisting of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone.
4. The kit for detecting multiple steroid hormones in biological fluids according to claim 1, further comprising: at least one of standard substance/standard substance working solution, quality control substance/quality control substance working solution, internal standard substance/internal standard substance working solution, matrix, eluent and double solvent.
5. The kit for detecting various steroids hormones in biological fluids according to claim 4, wherein the standard working solution is a standard methanol solution with a volume fraction of 50% to 100%;
the quality control product working solution is a quality control product methanol solution with the volume fraction of 50-100%;
the internal standard substance working solution is an internal standard substance methanol solution with the volume fraction of 50-100%;
the blank matrix comprises: blank plasma, serum, urine or saliva depleted of steroid hormones;
the eluent comprises: eluent 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, and eluent B is methanol containing formic acid with the volume fraction of 0.02-0.2%;
the double solvent comprises an aqueous solution of acetonitrile;
preferably, 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 internal standard is an isotopic internal standard for a steroid hormone;
the isotopic internal standard of steroid hormone comprises the isotope of steroid hormone13At least one of a C label and a deuterated label;
preferably, the internal standard comprises d 4-cortisol, d 8-cortisone, d 5-11-deoxycorticosterol, d 8-corticosterone, d 6-dehydroepiandrosterone, d 3-testosterone,13at least one of C3-17-hydroxyprogesterone, d 3-androstenedione, and d 9-progesterone.
6. The kit for detecting multiple steroid hormones in biological fluids according to claim 4, wherein the quality control substance working solution is a methanol solution containing multiple steroid hormones corresponding to the standard substance working solution, and comprises at least 1 concentration;
preferably, the standard working solution comprises at least 4 steroid hormone standard solutions of different concentrations; the quality control product working solution comprises at least 2 steroid hormone quality control product solutions with different concentrations.
7. The kit for detecting multiple steroid hormones in biological fluids according to claim 1, wherein said kit for detecting multiple steroid hormones in biological fluids comprises:
1) standard working solution: methanol solution containing cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone with volume fraction of 50-100%, with concentration of 4 or 5;
2) quality control product working solution: methanol solution containing cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone with volume fraction of 50-100%, with concentration of 2 or 3;
3) internal standard substance working solution: methanol solution containing 50-100% of cortisol, cortisone, 11-deoxycorticosterol, corticosterone, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone, androstenedione and progesterone isotope internal standard by volume fraction;
4) blank matrix: blank plasma, serum, urine or saliva depleted of steroid hormones;
5) liquid-liquid extracting agent: salting out at least one of an auxiliary liquid-liquid extraction reagent or tert-butyl methyl ether;
6) eluent: the eluent 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, and the eluent B is a methanol solution containing formic acid with the volume fraction of 0.02-0.2%.
8. A method for detecting multiple steroid hormones in biological fluids, which comprises the steps of using the kit for detecting multiple steroid hormones in biological fluids according to any one of claims 1 to 7, wherein the method comprises:
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.
9. The method for detecting multiple steroid hormones in a biological fluid as claimed in claim 8, wherein said method comprises:
step 1, pretreatment of a standard substance or quality control substance working solution: performing liquid-liquid extraction, centrifugation and redissolution on a mixed liquid of a standard substance or a quality control substance working solution and a corresponding isotope internal standard solution to obtain a standard substance to be detected;
step 2, pretreatment of the biological fluid sample: performing liquid-liquid extraction, centrifugation and redissolution on a mixed liquid of the biological fluid sample and a corresponding isotope internal standard liquid to obtain a sample to be detected;
step 3, detecting the sample to be detected by adopting a high performance liquid chromatography-tandem mass spectrometry method, and quantifying by adopting an isotope internal standard quantification method;
step 4, result analysis: and (3) taking the concentration of the standard working solution as an x axis, taking the peak area ratio of the standard working solution to the internal standard working solution as a y axis to draw a standard curve, and calculating the content of the steroid hormone in the biological fluid sample.
10. The method for detecting multiple steroid hormones in a biological fluid according to claim 9, wherein the amount of the isotope internal standard solution added is: the volume ratio of the biological body fluid to the isotope internal standard fluid is 1-15: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911285102.1A CN112964809A (en) | 2019-12-13 | 2019-12-13 | Kit for detecting multiple steroid hormones in biological body fluid and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911285102.1A CN112964809A (en) | 2019-12-13 | 2019-12-13 | Kit for detecting multiple steroid hormones in biological body fluid and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112964809A true CN112964809A (en) | 2021-06-15 |
Family
ID=76270793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911285102.1A Pending CN112964809A (en) | 2019-12-13 | 2019-12-13 | Kit for detecting multiple steroid hormones in biological body fluid and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112964809A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114674961A (en) * | 2022-04-13 | 2022-06-28 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof |
| CN115343398A (en) * | 2022-09-20 | 2022-11-15 | 吉林大学第一医院 | Kit and method for simultaneously measuring multiple steroid hormones in serum |
| CN116699003A (en) * | 2023-03-03 | 2023-09-05 | 中国民用航空局民用航空医学中心 | Circadian rhythm body fluid marker combination and UPLC-MS/MS detection method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807920A (en) * | 2015-05-21 | 2015-07-29 | 上海迪安医学检验所有限公司 | Kit for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique |
| US20160349221A1 (en) * | 2015-05-27 | 2016-12-01 | Quest Diagnostics Investments Llc | Methods for mass spectrometric quantitation of analytes extracted from a microsampling device |
| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN110187043A (en) * | 2019-04-25 | 2019-08-30 | 中南民族大学 | Method that is a kind of while detecting 13 kinds of steroid hormones in serum |
-
2019
- 2019-12-13 CN CN201911285102.1A patent/CN112964809A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807920A (en) * | 2015-05-21 | 2015-07-29 | 上海迪安医学检验所有限公司 | Kit for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique |
| US20160349221A1 (en) * | 2015-05-27 | 2016-12-01 | Quest Diagnostics Investments Llc | Methods for mass spectrometric quantitation of analytes extracted from a microsampling device |
| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN110187043A (en) * | 2019-04-25 | 2019-08-30 | 中南民族大学 | Method that is a kind of while detecting 13 kinds of steroid hormones in serum |
Non-Patent Citations (4)
| Title |
|---|
| CLARISSA DANIELA VOEGEL 等: "Steroid profiling in nails using liquid chromatography-tandem mass spectrometry" * |
| THERESE KOAL 等: "Standardized LC–MS/MS based steroid hormone profile-analysis", 《JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
| 徐玲燕 等: "LC-MS/MS在临床内源性类固醇激素分析中的方法研究和应用进展", 《药物分析杂志》 * |
| 米晓霞 等: "猪血浆中33种类固醇激素的高效液相色谱-串联质谱测定", 《分析测试学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114674961A (en) * | 2022-04-13 | 2022-06-28 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof |
| CN115343398A (en) * | 2022-09-20 | 2022-11-15 | 吉林大学第一医院 | Kit and method for simultaneously measuring multiple steroid hormones in serum |
| CN116699003A (en) * | 2023-03-03 | 2023-09-05 | 中国民用航空局民用航空医学中心 | Circadian rhythm body fluid marker combination and UPLC-MS/MS detection method thereof |
| CN116699003B (en) * | 2023-03-03 | 2023-12-26 | 中国民用航空局民用航空医学中心 | Circadian rhythm body fluid marker combination and UPLC-MS/MS detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111366671B (en) | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum | |
| McDonald et al. | Steroid profiling by gas chromatography–mass spectrometry and high performance liquid chromatography–mass spectrometry for adrenal diseases | |
| CN112964809A (en) | Kit for detecting multiple steroid hormones in biological body fluid and use method thereof | |
| CN109470791A (en) | A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol | |
| US20170328921A1 (en) | Methods for detecting hormones and other analytes | |
| CN114674961A (en) | Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof | |
| CN112611828B (en) | Method for purifying, enriching and detecting steroid hormone in blood | |
| CN110927289A (en) | Steroid hormone detection kit | |
| CN110702831B (en) | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry | |
| CN112611827B (en) | Chemical derivation-ultra high performance liquid chromatography-tandem mass spectrometry for detecting 3 estrogens | |
| CN110470753A (en) | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake | |
| CN111487328A (en) | Method for detecting multiple trace hormones in human body by mass spectrometry | |
| Davies et al. | Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry | |
| US11536733B2 (en) | Methods and systems for the detection of 11-oxo androgens by LC-MS/MS | |
| CN113720946A (en) | Method and kit for detecting multiple steroid hormones in blood | |
| Vicente et al. | Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry | |
| CN110531007B (en) | Detection method of arachidonic acid-like substance | |
| CN113009036A (en) | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones | |
| CN114636771B (en) | Method for detecting procaterol content in blood and application | |
| CN112964815A (en) | Method for detecting multiple steroid hormones in biological body fluid | |
| CN114705787A (en) | Method for detecting 12 steroid hormones in dry blood spots based on derivatization | |
| CN115343398A (en) | Kit and method for simultaneously measuring multiple steroid hormones in serum | |
| CN112557568B (en) | Method for detecting estradiol and estrone | |
| Yuan | A review on analysis of steroid profile in different biological matrices | |
| CN114609265A (en) | Method for detecting eight thyroid hormone markers in serum by liquid chromatography tandem mass spectrometry technology |
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 |