CN111487328A - Method for detecting multiple trace hormones in human body by mass spectrometry - Google Patents

Method for detecting multiple trace hormones in human body by mass spectrometry Download PDF

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CN111487328A
CN111487328A CN201910076824.XA CN201910076824A CN111487328A CN 111487328 A CN111487328 A CN 111487328A CN 201910076824 A CN201910076824 A CN 201910076824A CN 111487328 A CN111487328 A CN 111487328A
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solution
preparing
standard
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internal standard
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章天添
夏立凤
林奥
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Shanghai Goteway Biotechnology Group Co ltd
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Shanghai Goteway Biotechnology Group Co ltd
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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
    • 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
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • 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

Abstract

The invention discloses a method for detecting multiple trace hormones in a human body by using a mass spectrometry, belonging to the technical field of steroid hormone detection in serum. It comprises the following steps: (1) preparing a reagent; (2) preparing an extraction solvent; (3) preparing a standard stock solution; (4) preparing an internal standard substance stock solution; (5) preparing a standard curve solution; (6) processing a sample; (7) detecting a sample by a mass spectrometer; (8) and (4) determining the result by liquid chromatography/tandem mass spectrometry. There is no heterophile or autoantibody interference with this method; the structural analogue of hormone metabolism can be detected in a distinguishing way, and no cross reaction exists; the dynamic linear range is wide, and the hormone detection requirement with a large concentration span range can be met, for example, the situation that the concentration difference of the same hormone is large in different individuals or under different stress states of the same individual (testosterone and the like) can be met; the sensitivity of detecting the hormone with relatively low content is improved; can detect multiple hormones simultaneously.

Description

Method for detecting multiple trace hormones in human body by mass spectrometry
Technical Field
The invention relates to a method for detecting multiple trace hormones in a human body by using a mass spectrometry, belonging to the technical field of steroid hormone detection in serum.
Background
Trace hormones refer to hormones that are present in very small amounts in the serum, for example, steroid hormones such as testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, aldosterone, dehydroepiandrosterone, and pregnenolone in the serum. Steroid hormones are a class of trace, high-potency tetracyclic aliphatic hydrocarbon compounds secreted by the adrenal gland and gonads, having a cyclopentane-polyhydrophenanthrene parent nucleus. Has definite functions in maintaining life and regulating sexual function, development of organisms, immunoregulation, treatment of skin diseases and fertility control, and has important clinical significance and curative effect judgment significance in screening newborn and diagnosing endocrine diseases and related metabolic diseases.
The existing platform for accurately quantifying trace hormone in human body generally adopts radioimmunoassay, and the defects of the immunological method in hormone detection include: presence of heterophilic or autoantibody interference; structural analogs of hormone metabolism cannot be detected differentially, and cross reaction exists; the dynamic linear range is narrow, and the hormone detection requirement with a large concentration span range cannot be met, for example, the concentration difference of the same hormone in different individuals or under different stress states of the same individual is large (testosterone and the like); sensitivity to relatively low levels of hormone detection is insufficient; multiple hormones cannot be detected simultaneously. These drawbacks of immunological methods do not, to a certain extent, meet the need for a more comprehensive and accurate diagnosis in the field of endocrine diseases.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the method for detecting multiple trace hormones in a human body by using the mass spectrometry solves the problem that the detection result cannot meet the more comprehensive and accurate diagnosis requirement due to the defects of the conventional method for detecting the trace hormones by using an immunology method.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a method for detecting multiple trace hormones in a human body by mass spectrometry, comprising the steps of:
(1) preparing a reagent: preparing methanol (chromatographic grade), ammonium formate (chromatographic grade);
0.2 mol/L ammonium formate solution 1.26g ammonium formate was dissolved in water and the volume was 100m L;
absorbing 1m of L0.2 mol/L ammonium formate solution in L mmol of ammonium formate solution, adding the ammonium formate solution into water for dissolving, and fixing the volume to 100m of L;
absorbing 1m L/L of methanol ammonium formate solution, adding 0.2 mol/L of the methanol ammonium formate solution into methanol to dissolve the solution, and fixing the volume to 100m L;
(2) preparing an extraction solvent: preparing methyl tert-butyl ether (chromatographic grade), n-hexane (chromatographic grade), ethyl acetate (chromatographic grade) according to the weight ratio of n-hexane: ethyl acetate: preparing an extraction solvent from methyl tert-butyl ether (1: 1:1 by volume);
(3) preparing a standard stock solution: preparing 11 standard substances, namely testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, aldosterone, dehydroepiandrosterone and pregnenolone, wherein the purity is more than 97%, accurately weighing more than 10mg of the standard substances respectively, preparing a standard stock solution with a certain concentration by using methanol, and freezing and storing in a dark place at the temperature of-18 ℃;
(4) preparing an internal standard substance stock solution, namely preparing an isotope internal standard substance, testosterone-d 5, progesterone-d 9, 17-hydroxyprogesterone-d 8, aldosterone-d 7, cortisol-d 4, dehydroepiandrosterone-d 6, corticosterone-d 8 and corticosterone-d 8, preparing the internal standard stock solution of 0.1-1.0mg/m L by using methanol according to the capacity of the isotope internal standard substance, and freezing and storing at the temperature of-18 ℃ in a dark place;
(5) preparing a standard curve solution:
using standard stock solutions, internal standard substance stock solutions, to prepare linear concentration points of progesterone, 17-hydroxyprogesterone, androstenedione, corticosterone of 0.05, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L, linear concentration points of testosterone, dihydrotestosterone, corticosterone, aldosterone of 0.1, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L1, linear concentration points of cortisol of 1.0, 5.0, 10, 20, 50, 100, 200 mg/L, internal standard of 1.0 mg/L, to prepare linear concentration points of dehydroepiandrosterone, pregnenolone of 0.5, 1.0, 2.0, 5.0, 10, 20, 50 mg/L, internal standard of 10 mg/L, to draw up a suitable volume ratio of methanol to methanol at standard solution of 4835/5 mmol/5 mg/8 of ammonium formate (5: 2/5 mg/5 mmol/5 mg/2);
(6) sample treatment:
accurately transferring a serum sample to 400 mu L, adding 0.01 mg/L of an internal standard 20 mu L0 when measuring testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone and aldosterone 9 steroid hormones, adding 0.1 mg/L of the internal standard 20 mu L when measuring dehydroepiandrosterone and pregnenolone 2 steroid hormones, whirling for 30s, adding an extraction solvent 1.0m L, whirling for 3min, centrifuging for 5min at 4000r/min, taking 0.9m L clear liquid into another centrifuge tube, adding 0.8m L extraction solution, repeating once, combining two extraction solutions, drying nitrogen, fixing the volume to 0.2m L by using 2 mmol/L of aqueous solution of 2 mmol/L of methanol-2: 8 (volume ratio), and loading on a machine for testing;
(7) detecting a sample by a mass spectrometer:
chromatographic conditions are as follows:
a. chromatographic column CORTECS-C18 of 2.7 μm and 100mm × 2.1.1 mm;
b. mobile phase A is 2 mmol/L ammonium formate aqueous solution, and mobile phase B is 2 mmol/L ammonium formate methanol solution;
c. the flow rate is 0.4m L/min;
d. column temperature: 40 ℃;
e. the sample injection amount is 20u L;
mass spectrum conditions:
a. an ion source: an electrospray ion source;
b. the scanning mode is as follows: scanning positive ions;
c. the detection mode is as follows: monitoring multiple reactions;
d.IonSpray Voltage：5500V；
e.Temperature：600℃；
f.Curtain Gas：25μL/min；
g.Collision Gas：7μL/min；
h.Ion Source Gas 1：50μL/min；
i.Ion Source Gas 2：55μL/min；
j. qualitative ion pairs, quantitative ion pairs, collision energy and declustering voltage;
measuring the qualitative ion pair, the quantitative ion pair, the collision gas energy and the declustering voltage of the 11 steroid hormone solutions by the mass spectrometer set under the conditions;
(8) liquid chromatography/tandem mass spectrometry results:
a. qualitative determination
Determining the sample solution and the standard solution according to the steps, if the retention time of the mass chromatographic peak of the sample solution is consistent with that of the standard solution; the relative abundance of the qualitative ion pair is consistent with that of the mixed solution with a corresponding concentration, and the deviation of the relative abundance does not exceed the maximum allowable deviation of the relative abundance of the ions in the qualitative determination, so that the corresponding measured object in the sample can be judged;
b. quantitative determination
After the instrument is stabilized, the sample solution and the standard solution are injected with equal volume, a standard working curve is drawn, an internal standard method is adopted to carry out quantitative calculation on the sample, and the response value of the analyte in the sample solution is within the linear range measured by the instrument, so that a quantitative result is obtained.
And (3) quantitative calculation:
the measurement result is automatically calculated by the instrument workstation according to an internal standard method.
The steroid hormone content of the sample was calculated as follows:
X＝Ci×V2/V1
in the formula:
x- -the amount of hormone in the sample in nanograms per kilogram (ng/m L);
ci- -concentration of steroid hormone in sample preparation in nanograms per milliliter (ng/m L);
v2 — final volumetric volume in milliliters (m L);
v1- -volume of sample in milliliters (m L).
The method has the following quantitative limit:
the method has the detection limits of 0.01ng/m L for progesterone, 17-hydroxyprogesterone, androstenedione and corticosterone, the quantification limit of 0.025ng/m L, the detection limits of 0.025ng/m L for testosterone, dihydrotestosterone, cortisol, corticosterone and aldosterone, the quantification limit of 0.05ng/m L, the detection limits of 0.10ng/m L for dehydroepiandrosterone and pregnenolone, and the quantification limit of 0.25ng/m L.
And (3) recovery rate:
when the adding concentration of the method is 0.025ng/m L-5.0 ng/m L, the recovery rate is 80-110%.
The invention has the beneficial effects that:
(1) absence of heterophilic or autoantibody interference;
(2) the structural analogue of hormone metabolism can be detected in a distinguishing way, and no cross reaction exists;
(3) the dynamic linear range is wide, and the hormone detection requirement with a large concentration span range can be met, for example, the situation that the concentration difference of the same hormone is large in different individuals or under different stress states of the same individual (testosterone and the like) can be met;
(4) the sensitivity of detecting the hormone with relatively low content is improved;
(5) can detect multiple hormones simultaneously.
Adding an internal standard into a serum sample, extracting by using a mixed solution of methyl tert-butyl ether, normal hexane and ethyl acetate, and fixing the volume by using ammonium formate and a methanol solution after nitrogen blowing; liquid chromatogram-tandem mass spectrometer determination, multi-reaction ion monitoring and detection, and internal standard method quantification; .
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below.
A method for detecting multiple trace hormones in a human body by mass spectrometry, comprising the steps of:
(1) preparing a reagent: preparing methanol (chromatographic grade), ammonium formate (chromatographic grade);
0.2 mol/L ammonium formate solution 1.26g ammonium formate was dissolved in water and the volume was 100m L;
absorbing 1m of L0.2 mol/L ammonium formate solution in L mmol of ammonium formate solution, adding the ammonium formate solution into water for dissolving, and fixing the volume to 100m of L;
absorbing 1m L/L of methanol ammonium formate solution, adding 0.2 mol/L of the methanol ammonium formate solution into methanol to dissolve the solution, and fixing the volume to 100m L;
(2) preparing an extraction solvent: preparing methyl tert-butyl ether (chromatographic grade), n-hexane (chromatographic grade), ethyl acetate (chromatographic grade) according to the weight ratio of n-hexane: ethyl acetate: preparing an extraction solvent from methyl tert-butyl ether (1: 1:1 by volume);
(3) preparing a standard stock solution: preparing 11 standard substances, namely testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, aldosterone, dehydroepiandrosterone and pregnenolone, wherein the purity is more than 97%, accurately weighing more than 10mg of the standard substances respectively, preparing a standard stock solution with a certain concentration by using methanol, and freezing and storing in a dark place at the temperature of-18 ℃;
(4) preparing an internal standard substance stock solution, namely preparing an isotope internal standard substance, testosterone-d 5, progesterone-d 9, 17-hydroxyprogesterone-d 8, aldosterone-d 7, cortisol-d 4, dehydroepiandrosterone-d 6, corticosterone-d 8 and corticosterone-d 8, preparing the internal standard stock solution of 0.1-1.0mg/m L by using methanol according to the capacity of the isotope internal standard substance, and freezing and storing at the temperature of-18 ℃ in a dark place;
(5) preparing a standard curve solution:
using standard stock solutions, internal standard substance stock solutions, to prepare linear concentration points of progesterone, 17-hydroxyprogesterone, androstenedione, corticosterone of 0.05, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L, linear concentration points of testosterone, dihydrotestosterone, corticosterone, aldosterone of 0.1, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L1, linear concentration points of cortisol of 1.0, 5.0, 10, 20, 50, 100, 200 mg/L, internal standard of 1.0 mg/L, to prepare linear concentration points of dehydroepiandrosterone, pregnenolone of 0.5, 1.0, 2.0, 5.0, 10, 20, 50 mg/L, internal standard of 10 mg/L, to draw up a suitable volume ratio of methanol to methanol at standard solution of 4835/5 mmol/5 mg/8 of ammonium formate (5: 2/5 mg/5 mmol/5 mg/2);
(6) sample treatment:
accurately transferring a serum sample to 400 mu L, adding 0.01 mg/L of an internal standard 20 mu L0 when measuring testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone and aldosterone 9 steroid hormones, adding 0.1 mg/L of the internal standard 20 mu L when measuring dehydroepiandrosterone and pregnenolone 2 steroid hormones, whirling for 30s, adding an extraction solvent 1.0m L, whirling for 3min, centrifuging for 5min at 4000r/min, taking 0.9m L clear liquid into another centrifuge tube, adding 0.8m L extraction solution, repeating once, combining two extraction solutions, drying nitrogen, fixing the volume to 0.2m L by using 2 mmol/L of aqueous solution of 2 mmol/L of methanol-2: 8 (volume ratio), and loading on a machine for testing;
(7) detecting a sample by a mass spectrometer:
chromatographic conditions are as follows:
a. chromatographic column CORTECS-C18 of 2.7 μm and 100mm × 2.1.1 mm;
b. mobile phase A is 2 mmol/L ammonium formate aqueous solution, mobile phase B is 2 mmol/L ammonium formate methanol solution, and mobile phase gradient (see table I);
c. the flow rate is 0.4m L/min;
d. column temperature: 40 ℃;
e. the sample injection amount is 20u L;
meter-mobile phase gradiometer
Figure BDA0001959120490000071
Figure BDA0001959120490000081
Mass spectrum conditions:
a. an ion source: an electrospray ion source;
b. the scanning mode is as follows: scanning positive ions;
c. the detection mode is as follows: monitoring multiple reactions;
d.IonSpray Voltage：5500V；
e.Temperature：600℃；
f.Curtain Gas：25μL/min；
g.Collision Gas：7μL/min；
h.Ion Source Gas 1：50μL/min；
i.Ion Source Gas 2：55μL/min；
j. qualitative ion pairs, quantitative ion pairs, collision energy and declustering voltage (see table two);
measuring the qualitative ion pair, the quantitative ion pair, the collision gas energy and the declustering voltage of the 11 steroid hormone solutions by the mass spectrometer set under the conditions;
TABLE 11 qualitative ion-pair, quantitative ion-pair, collisional gas energy and declustering voltage of steroid hormones
Figure BDA0001959120490000082
Figure BDA0001959120490000091
Figure BDA0001959120490000101
(8) Liquid chromatography/tandem mass spectrometry results:
a. qualitative determination
Determining the sample solution and the standard solution according to the steps, if the retention time of the mass chromatographic peak of the sample solution is consistent with that of the standard solution; the relative abundance of the qualitative ion pair is consistent with that of the mixed solution with a corresponding concentration, and the deviation of the relative abundance does not exceed the maximum allowable deviation of the relative abundance of the ions during qualitative determination (see table III), so that the corresponding measured object in the sample can be judged;
maximum allowable deviation of ion relative abundance in the case of TABLE III qualitative determination
Relative ion abundance >50％ >20～50％ >10～20％ ≤10％
Allowable relative deviation ±20％ ±25％ ±30％ ±50％
b. Quantitative determination
After the instrument is stabilized, the sample solution and the standard solution are injected with equal volume, a standard working curve is drawn, an internal standard method is adopted to carry out quantitative calculation on the sample, and the response value of the analyte in the sample solution is within the linear range measured by the instrument, so that a quantitative result is obtained.
And (3) quantitative calculation:
the measurement result is automatically calculated by the instrument workstation according to an internal standard method.
The steroid hormone content of the sample was calculated as follows:
X＝Ci×V2/V1
in the formula:
x- -the amount of hormone in the sample in nanograms per kilogram (ng/m L);
ci- -concentration of steroid hormone in sample preparation in nanograms per milliliter (ng/m L);
v2 — final volumetric volume in milliliters (m L);
v1- -volume of sample in milliliters (m L).
The method has the following quantitative limit:
the method has the detection limits of 0.01ng/m L for progesterone, 17-hydroxyprogesterone, androstenedione and corticosterone, the quantification limit of 0.025ng/m L, the detection limits of 0.025ng/m L for testosterone, dihydrotestosterone, cortisol, corticosterone and aldosterone, the quantification limit of 0.05ng/m L, the detection limits of 0.10ng/m L for dehydroepiandrosterone and pregnenolone, and the quantification limit of 0.25ng/m L.
And (3) recovery rate:
when the adding concentration of the method is 0.025ng/m L-5.0 ng/m L, the recovery rate is 80-110%.
Adding an internal standard into a serum sample, extracting by using a mixed solution of methyl tert-butyl ether, normal hexane and ethyl acetate, and fixing the volume by using ammonium formate and a methanol solution after nitrogen blowing; liquid chromatogram-tandem mass spectrometer determination, multi-reaction ion monitoring and detection, and internal standard method quantification; there is no heterophile or autoantibody interference with this method; the structural analogue of hormone metabolism can be detected in a distinguishing way, and no cross reaction exists; the dynamic linear range is wide, and the hormone detection requirement with a large concentration span range can be met, for example, the situation that the concentration difference of the same hormone is large in different individuals or under different stress states of the same individual (testosterone and the like) can be met; the sensitivity of detecting the hormone with relatively low content is improved; can detect multiple hormones simultaneously.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (1)

1. A method for detecting a plurality of trace hormones in a human body by mass spectrometry, which is characterized by comprising the following steps:
(1) preparing a reagent: preparing methanol (chromatographic grade), ammonium formate (chromatographic grade);
0.2 mol/L ammonium formate solution 1.26g ammonium formate was dissolved in water and the volume was 100m L;
absorbing 1m of L0.2 mol/L ammonium formate solution in L mmol of ammonium formate solution, adding the ammonium formate solution into water for dissolving, and fixing the volume to 100m of L;
absorbing 1m L/L of methanol ammonium formate solution, adding 0.2 mol/L of the methanol ammonium formate solution into methanol to dissolve the solution, and fixing the volume to 100m L;
(2) preparing an extraction solvent: preparing methyl tert-butyl ether (chromatographic grade), n-hexane (chromatographic grade), ethyl acetate (chromatographic grade) according to the weight ratio of n-hexane: ethyl acetate: preparing an extraction solvent from methyl tert-butyl ether (1: 1:1 by volume);
(3) preparing a standard stock solution: preparing 11 standard substances, namely testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, aldosterone, dehydroepiandrosterone and pregnenolone, wherein the purity is more than 97%, accurately weighing more than 10mg of the standard substances respectively, preparing a standard stock solution with a certain concentration by using methanol, and freezing and storing in a dark place at the temperature of-18 ℃;
(4) preparing an internal standard substance stock solution, namely preparing an isotope internal standard substance, testosterone-d 5, progesterone-d 9, 17-hydroxyprogesterone-d 8, aldosterone-d 7, cortisol-d 4, dehydroepiandrosterone-d 6, corticosterone-d 8 and corticosterone-d 8, preparing the internal standard stock solution of 0.1-1.0mg/m L by using methanol according to the capacity of the isotope internal standard substance, and freezing and storing at the temperature of-18 ℃ in a dark place;
(5) preparing a standard curve solution:
using standard stock solutions, internal standard substance stock solutions, to prepare linear concentration points of progesterone, 17-hydroxyprogesterone, androstenedione, corticosterone of 0.05, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L, linear concentration points of testosterone, dihydrotestosterone, corticosterone, aldosterone of 0.1, 0.2, 0.5, 2.0, 5.0, 10, 20 mg/L, internal standard of 1.0 mg/L1, linear concentration points of cortisol of 1.0, 5.0, 10, 20, 50, 100, 200 mg/L, internal standard of 1.0 mg/L, to prepare linear concentration points of dehydroepiandrosterone, pregnenolone of 0.5, 1.0, 2.0, 5.0, 10, 20, 50 mg/L, internal standard of 10 mg/L, to draw up a suitable volume ratio of methanol to methanol at standard solution of 4835/5 mmol/5 mg/8 of ammonium formate (5: 2/5 mg/5 mmol/5 mg/2);
(6) sample treatment:
accurately transferring a serum sample to 400 mu L, adding 0.01 mg/L of an internal standard 20 mu L0 when measuring testosterone, androstenedione, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, cortisol, corticosterone and aldosterone 9 steroid hormones, adding 0.1 mg/L of the internal standard 20 mu L when measuring dehydroepiandrosterone and pregnenolone 2 steroid hormones, whirling for 30s, adding an extraction solvent 1.0m L, whirling for 3min, centrifuging for 5min at 4000r/min, taking 0.9m L clear liquid into another centrifuge tube, adding 0.8m L extraction solution, repeating once, combining two extraction solutions, drying nitrogen, fixing the volume to 0.2m L by using 2 mmol/L of aqueous solution of 2 mmol/L of methanol-2: 8 (volume ratio), and loading on a machine for testing;
(7) detecting a sample by a mass spectrometer:
chromatographic conditions are as follows:
a. chromatographic column CORTECS-C18 of 2.7 μm and 100mm × 2.1.1 mm;
b. mobile phase A is 2 mmol/L ammonium formate aqueous solution, and mobile phase B is 2 mmol/L ammonium formate methanol solution;
c. the flow rate is 0.4m L/min;
d. column temperature: 40 ℃;
e. the sample injection amount is 20u L;
mass spectrum conditions:
a. an ion source: an electrospray ion source;
b. the scanning mode is as follows: scanning positive ions;
c. the detection mode is as follows: monitoring multiple reactions;
d.IonSpray Voltage：5500V；
e.Temperature：600℃；
f.Curtain Gas：25μL/min；
g.Collision Gas：7μL/min；
h.Ion Source Gas 1：50μL/min；
i.Ion Source Gas 2：55μL/min；
j. qualitative ion pairs, quantitative ion pairs, collision energy and declustering voltage;
measuring the qualitative ion pair, the quantitative ion pair, the collision gas energy and the declustering voltage of the 11 steroid hormone solutions by the mass spectrometer set under the conditions;
(8) liquid chromatography/tandem mass spectrometry results:
a. qualitative determination
Determining the sample solution and the standard solution according to the steps, if the retention time of the mass chromatographic peak of the sample solution is consistent with that of the standard solution; the relative abundance of the qualitative ion pair is consistent with that of the mixed solution with a corresponding concentration, and the deviation of the relative abundance does not exceed the maximum allowable deviation of the relative abundance of the ions in the qualitative determination, so that the corresponding measured object in the sample can be judged;
b. quantitative determination
After the instrument is stabilized, the sample solution and the standard solution are injected with equal volume, a standard working curve is drawn, an internal standard method is adopted to carry out quantitative calculation on the sample, and the response value of the analyte in the sample solution is within the linear range measured by the instrument, so that a quantitative result is obtained.
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CN113009006A (en) * 2021-01-20 2021-06-22 泊迈生物医学检测(苏州)有限公司 Method and kit for detecting dehydroepiandrosterone content in saliva
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CN114487185A (en) * 2022-01-21 2022-05-13 武汉迈特维尔生物科技有限公司 Separation and identification method of cholesterol pathway
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
CN115236246A (en) * 2022-09-26 2022-10-25 北京金域医学检验实验室有限公司 Method for extracting multiple steroid hormones from organism fluid sample

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