CN113720946A - Method and kit for detecting multiple steroid hormones in blood - Google Patents

Abstract

The invention discloses a method for detecting multiple steroid hormones in blood, which comprises the steps of detecting DHEA, DHT, P5 and 17OHP5 in the blood after pretreatment by adopting a high performance liquid chromatography-tandem mass spectrometry technology; s1, separating DHEA, DHT, P5 and 17OHP5 from interferents by using high performance liquid chromatography; s2, quantifying by using an isotope internal standard method; and S3, establishing a standard curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of DHEA, DHT, P5 and 17OHP5 in the sample to be detected. A kit comprising the following reagents: a mobile phase comprising mobile phase A and mobile phase B; mobile phase A: 0.1% by volume of formic acid aqueous solution; mobile phase B: acetonitrile, chromatographic purity. The invention has strong specificity, high sensitivity, simple pretreatment process, precision and standard recovery rate basically meeting the requirements, and can be used for analyzing the content of 4 steroid hormones with low ionization efficiency in blood.

Description

Method and kit for detecting multiple steroid hormones in blood

Technical Field

The invention relates to the technical field of biological detection, in particular to a method and a kit for detecting multiple steroid hormones in blood.

Background

Steroid hormones, also known as steroid hormones, are tetracyclic aliphatic hydrocarbon compounds having a cyclopentane-polyhydrophenanthrene nucleus. Human steroid hormones are mainly classified into two major classes, adrenocortical hormones and sex hormones, and have important physiological roles in the aspects of human function development, metabolic cycle and the like.

Generally, an immunological method is generally used for detecting the content of the steroid in blood, but the immunological method is easily interfered by endogenous structural analogues to generate cross reaction when analyzing small molecular compounds, so that the detection result is unexpectedly deviated. In recent years, the liquid chromatography tandem mass spectrometry technology is more and more widely applied to the analysis and detection of human endogenous steroid hormones due to the advantages of high sensitivity, high specificity and the like. However, when some steroid hormones are analyzed by using a liquid chromatography tandem mass spectrometry technology, the ionization efficiency of molecules of the steroid hormones is low, so that the detection sensitivity is low and the conventional detection requirements cannot be met.

To solve the above problems, we propose a method and a kit for detecting various steroid hormones in blood.

Disclosure of Invention

The invention aims to solve the problem that the detection of steroid hormone in 4 in blood is not ideal in the prior art, and provides a method and a kit for detecting multiple steroid hormones in blood.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting multiple steroid hormones in blood comprises detecting DHEA, DHT, P5, and 17OHP5 in pretreated blood by high performance liquid chromatography-tandem mass spectrometry;

s1, separating DHEA, DHT, P5 and 17OHP5 from interferents by using high performance liquid chromatography;

s2, quantifying by using an isotope internal standard method;

and S3, establishing a standard curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of DHEA, DHT, P5 and 17OHP5 in the sample to be detected.

Preferably, the specific conditions of the high performance liquid chromatography are as follows:

mobile phase A: formic acid water with the volume ratio of 0.1 percent;

mobile phase B: acetonitrile with purity of chromatographic purity; the concentration is 100%;

the type of the chromatographic column: waters XBridge BEH C18, 3.5 μm,2.1mm 150 mm;

a gradient elution mode is adopted, and the method is shown in a table 1;

the flow rate was 0.2ml/min, the column temperature was 40 ℃ and the injection volume was 10. mu.l.

TABLE 1 mobile phase gradient elution parameters

Preferably, the mass spectrometry conditions are:

under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 5500V; the collision gas is 8; the air curtain air is 20 psi; the ion source temperature is 650 ℃; ion source gas stream GS1 was 70 psi; GS2 at 50 psi; target DHEA m/z 332.2 → 253.1, d6-DHEA m/z 338.2 → 253.1; DHT m/z 334.2 → 105.1, d3-T m/z 337.2 → 105.1; p5m/z 360.2 → 300.1, d2,¹³C2-P5 m/z 364.2→304.1；17OHP5 m/z 358.2→145.1，d2,¹³C2-17OHP5 m/z 362.2→145.1；

the 5 steroid hormones and the internal standard declustering voltage-DP, injection voltage-EP, collision voltage-CE, collision chamber injection voltage-CXP parameters are shown in Table 2.

TABLE 2 Mass Spectrometry parameters of Testosterone and internal standards

Preferably, the pretreated blood is prepared according to the following method: adding 300 mu l of blood into a 2ml centrifuge tube, adding the mixed internal standard solution, carrying out vortex oscillation for 1min, centrifuging for 10min at 13000r/min, transferring all clear liquid to another 2ml centrifuge tube, carrying out nitrogen blow-drying, adding water into the blow-dried centrifuge tube, slightly carrying out vortex oscillation, adding the extract liquid, carrying out vortex oscillation extraction, transferring an organic phase to another 2ml centrifuge tube, repeating the operation, carrying out extraction once again, and combining the two organic phases; drying by nitrogen;

adding a derivatization agent into the dried centrifugal tube, and sealing and avoiding light for water bath reaction; after the reaction is finished, centrifuging at 13000r/min for 5min, taking the supernatant, and sampling 10 mul to test on a machine.

Preferably, the internal standard solution is mixed to prepare: accurately weighing 1mg of d6-DHEA, d3-DHT and d2 respectively,¹³C2-P5、d2,¹³dissolving C2-17OHP5 standard substance in 4 10ml volumetric flasks, dissolving with acetonitrile to constant volume to obtain d6-DHEA, d3-DHT and d2 with the concentration of 100 mu g/ml,¹³C2-P5、d2,¹³c2-17OHP5 internal standard mother liquor;

taking 10 mul of d6-DHEA and d2 with the concentration of 100 mug/ml,¹³C2-P5、d2,¹³c2-17OHP5 and 1 mul 100 mug/ml d3-DHT internal standard mother liquor are put into a 10ml volumetric flask, diluted by acetonitrile to be calibrated to obtain 100ng/ml d6-DHEA and d2,¹³C2-P5、d2,¹³c2-17OHP5 and 10ng/ml d3-DHT mixed solution;

diluting 0.25ml of the mixed solution with acetonitrile to 100ml to obtain d6-DHEA and d2 with the concentration of 0.25ng/ml,¹³C2-P5、d2,¹³c2-17OHP5 and 0.025ng/ml d3-DHT mixed internal standard solution for experiments.

Preferably, the volume of the added mixed internal standard solution is 1ml, the blow-drying temperature of nitrogen is 60 ℃, the volume of water added into a blow-dried centrifugal tube is 0.3ml, the extract is methyl tert-butyl ether, the volume of the extract is 1ml, the vortex oscillation extraction time is 10min, the ethoxyamine hydrochloride with the derivatization agent concentration of 100mmol/l is added, the solvent is 60% methanol water, and the volume of the derivatization agent is 100 mul; the reaction temperature of the lucifugal water bath is 60 ℃, and the reaction time is 60 min.

A kit comprising the following reagents:

a mobile phase comprising mobile phase A and mobile phase B;

mobile phase A: 0.1% by volume of formic acid aqueous solution;

mobile phase B: acetonitrile, chromatographic purity;

and (3) standard mother liquor: a solution of dehydroepiandrosterone, dihydrotestosterone, pregnenolone, 17-hydroxypregnenolone in acetonitrile;

internal standard solution: d 6-dehydroepiandrosterone, d 3-testosterone, d2,¹³c2-pregnenolone, d2,¹³acetonitrile solution of C2-17-hydroxypregnanolone;

diluting liquid: comprises a first diluent and a second diluent;

diluting a first solution: a blank serum matrix solution, which is prepared by dissolving 1g of bovine serum albumin with water and fixing the volume to 100 ml;

and (2) diluting a second solution: acetonitrile;

extracting liquid: methyl tert-butyl ether;

quality control product: the blank serum matrix solution of dehydroepiandrosterone, pregnenolone and 17-hydroxypregnenolone is divided into low QCL, medium QCM and high QCH concentrations, and the concentrations are respectively as follows: 0.3ng/ml, 3ng/ml and 8 ng/ml; the blank serum matrix solution of dihydrotestosterone is divided into low QCL, medium QCM and high QCH concentrations, and the concentrations are respectively as follows: 0.03ng/ml, 0.3ng/ml and 0.8 ng/ml;

respectively taking 10 mu l of dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-hydroxypregnenolone standard mother liquor in a 10ml volumetric flask, diluting the solution to a certain volume by using a diluent to obtain a mixed standard solution of 1 mu g/ml dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone and 0.1 mu g/ml dihydrotestosterone;

preparing blank serum matrix solution of dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-hydroxypregnenolone:

low QCL: taking 3 mul of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

medium QCM: taking 30 mu l of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

high QCH: taking 80 mul of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

redissolving the solvent: 65% acetonitrile water solution by volume.

Preferably, preparing a standard mother solution: respectively and accurately weighing 10mg of dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone standard substance and 1mg of dihydrotestosterone standard substance in 4 10ml volumetric flasks, dissolving with acetonitrile and fixing the volume to a scale to obtain 1mg/ml of dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone standard substance mother liquor and 0.1mg/ml of dihydrotestosterone standard substance mother liquor;

preparing an internal standard solution: accurately weighing 1mg of d 6-dehydroepiandrosterone, d 3-testosterone and d2 respectively,¹³c2-pregnenolone, d2,¹³dissolving C2-17-hydroxypregnenolone standard in 4 10ml volumetric flasks, dissolving with acetonitrile, metering to desired volume to obtain d 6-dehydroepiandrosterone, d 3-testosterone, and d2 at a concentration of 100 μ g/ml,¹³c2-pregnenolone, d2,¹³c2-17-hydroxypregnanolone internal standard mother liquor.

Compared with the prior art, the invention has the beneficial effects (aiming at the technical problem) that:

1. the content of 4 steroid hormones (dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-alpha-hydroxypregnenolone) in blood is measured by adopting a liquid chromatography-tandem mass spectrometry technology in combination with an isotope dilution method. Compared with the defects of low sensitivity and poor specificity of the traditional immunoassay method, the method has the advantages that the steroid hormone and the ethoxyamine react to generate the oxime ester, the detection sensitivity is obviously improved, the specificity of the detection method is ensured by adopting a reaction detection mode (MRM), and in addition, the potential interference and the matrix effect are well eliminated and corrected by the online chromatographic separation and the application of an isotope internal standard substance.

2. The methodology of the method is verified, and analysis after treatment with the blank matrix solution shows that the method has high specificity and has no obvious interference at the positions of the target substance and the internal standard substance. And (3) precision test results: the precision range in the day is 1.41% -8.53%; the precision range in daytime is 0.73% -8.53%, and the method meets the acceptance standard (CV is less than or equal to 15%), which shows that the method has good stability. The standard adding recovery rate test is carried out by adding 3 standard substances with low, medium and high concentrations, and the result shows that the standard adding recovery rate of the 4 steroid hormones is between 84.0 and 113.3 percent and meets the acceptable standard (80 to 120 percent).

In conclusion, the method has high sensitivity and good specificity, the blood sample is simpler to process, and the methodological verification meets the requirements, which shows that the method is a reliable quantitative detection method which can be used for Dehydroepiandrosterone (DHEA), Dihydrotestosterone (DHT), pregnenolone (P5) and 17-alpha-hydroxypregnenolone (17OHP5) in human blood.

Drawings

FIG. 1 is an ion chromatogram for extraction of 4 kinds of steroid hormones from a standard substance of steroid hormones in blood according to the method for detecting multiple kinds of steroid hormones in blood of the present invention;

FIG. 2 is a schematic structural diagram of an ion chromatogram for extraction of 4 kinds of steroid hormones from a blood sample in the method for detecting multiple kinds of steroid hormones in blood according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Referring to FIGS. 1-2, a method for detecting multiple steroid hormones in blood;

methodology samples for the study experiments were obtained from blood samples from employees of the echotaceae biotechnology (Jiangsu) Co.

The instrument comprises the following steps: API 5500 triple quadrupole mass spectrometer (AB SCIEX corporation); LC-20AD XR liquid chromatography system (Shimadzu Corp.); temperature controlled high speed centrifuge (Eppendorf corporation); ultra pure water instruments (Millipore corporation); multi-tube vortex oscillators (hang hou ruicheng instruments ltd); nitrogen blowing instruments (hang state ruicheng instruments ltd); vortex oscillators (available from great-dragon, beijing, inc.); a water bath (Shanghai-Hengscientific instruments Co., Ltd.); a pipette (Eppendorf Co., 0.5-10. mu.l, 20-200. mu.l, 100-1000. mu.l), a precision balance (Beijing Saedodes scientific instruments Co., Ltd.), and the like.

Reagent consumables: chromatographic grade acetonitrile, formic acid (Sigma-Aldrich), Waters XBridge BEH C18 reverse phase chromatography column (3.5 μm,2.1mm 150mm, Waters); chromatographically pure methyl tert-butyl ether (Sigma-Aldrich), ethoxyamine hydrochloride (Sigma-Aldrich); bovine serum albumin (purity 99% or more, Sigma-Aldrich Co.).

And (3) standard substance: the DHEA, DHT, P5 and 17OHP5 standard substances are all purchased from Torto Research Chemicals Inc., and the purity is more than or equal to 99 percent; d6-DHEA, d3-DHT, d2,13C2-P5, d2 and 13C2-17OHP5 are all purchased from Cambridge Isotrope Laboratories Inc., and the purity is more than or equal to 98 percent.

Quality control product: blank serum matrix solution containing low, medium and high 3 concentrations of 5 steroid hormones. DHEA, P5 and 17OHP5 low-concentration quality control QC (L), medium-concentration quality control QC (M) and high-concentration quality control QC (H), wherein the concentrations are 0.3ng/ml, 3ng/ml and 8ng/ml respectively; DHT low concentration quality control QC (L), medium concentration quality control QC (M), and high concentration quality control QC (H), with concentrations of 0.03ng/ml, 0.3ng/ml, and 0.8ng/ml, respectively.

Method

Chromatographic conditions are as follows: mobile phase A: formic acid water with the volume ratio of 0.1 percent; mobile phase B: acetonitrile, concentration 100%. The type of the chromatographic column: waters Xbridge BEH C18(3.5 μm,2.1 mm. times.150 mm). A gradient elution mode was used, as detailed in Table 1, with a flow rate of 0.2ml/min, a column temperature of 40 ℃ and a sample injection volume of 10. mu.l.

Mass spectrum conditions: adopting a mass spectrum scanning mode of multi-reaction monitoring (MRM) in an electrospray ionization positive ion detection mode; the spraying voltage is 5500V; the collision gas is 8; the air curtain air is 20 psi; the ion source temperature is 650 ℃; ion source gas stream GS1 was 70 psi; GS2 was 50 psi. The parameters of 5 steroid hormones, declustering voltage (DP), injection voltage (EP), Collision Energy (CE), and collision cell outlet voltage (CXP) of the corresponding internal standard are set up in detail in Table 2.

Preparing a standard substance:

respectively and accurately weighing 10mg of DHEA, P5, 17OHP5 standard substance and 1mg of DHT standard substance in 4 10ml volumetric flasks, dissolving with acetonitrile and fixing the volume to a scale to obtain 1mg/ml of DHEA, P5, 17OHP5 standard substance mother liquor and 0.1mg/ml of DHT standard substance mother liquor; respectively taking 10 mu l of DHEA, P5, 17OHP5 and DHT standard mother liquor in a 10ml volumetric flask, and diluting to a constant volume with a diluent 2 to obtain a mixed standard solution of 1 mu g/ml DHEA, P5, 17OHP5 and 0.1 mu g/ml DHT; 100 μ l of the mixed standard solution is taken and diluted to 10ml by the diluent 1, and the mixed standard solution of 10ng/ml DHEA, P5, 17OHP5 and 1ng/ml DHT is obtained.

1mg of d6-DHEA, d3-DHT, d2,13C2-P5, d2 and 13C2-17OHP5 standard substances are accurately weighed in 4 10ml volumetric flasks respectively, and are dissolved by acetonitrile to be constant volume to scale, so that internal standard mother liquor of 100 mu g/ml of d6-DHEA, d3-DHT, d2,13C2-P5, d2 and 13C 2-OHP 5 is obtained. Taking 10 mu l of 100 mu g/ml d6-DHEA, d2,13C2-P5, d2,13C2-17OHP5 and 1 mu l of 100 mu g/ml d3-DHT internal standard mother liquor to a 10ml volumetric flask, diluting with acetonitrile to a constant volume to obtain 100ng/ml d6-DHEA, d2,13C2-P5, d2,13C2-17OHP5 and 10ng/ml d3-DHT internal standard mixed solution. 0.25ml of the internal standard mixed solution is diluted by acetonitrile to be 100ml to obtain the experimental internal standard mixed solution with the concentration of 0.25ng/ml d6-DHEA, d2,13C2-P5, d2,13C2-17OHP5 and 0.025ng/ml d 3-DHT.

Preparation of quality control product

The blank serum matrix solution of DHEA, P5 and 17OHP5 has low QC (L), medium QC (M) and high QC (H) concentrations respectively as follows: 0.3ng/ml, 3ng/ml and 8 ng/ml; the blank serum matrix solution of DHT was divided into low QC (L), medium QC (M), and high QC (H) concentrations, which were: 0.03ng/ml, 0.3ng/ml and 0.8 ng/ml;

respectively taking 10 mu l of mother liquor of the DHEA, DHT, P5 and 17OHP5 standard substances in a 10ml volumetric flask, and fixing the volume to the scale by using a diluent 2 to obtain a mixed standard solution of 1 mu g/ml DHEA, P5, 17OHP5 and 0.1 mu g/ml DHT;

blank serum base solution preparation of DHEA, DHT, P5, 17OHP 5:

QC (L): taking 3 mul of the mixed standard solution, and fixing the volume to 10ml by using the diluent 1;

QC (M): taking 30 mul of the mixed standard solution, and fixing the volume to 10ml by using the diluent 1;

QC (H): taking 80 mul of the mixed standard solution, and fixing the volume to 10ml by using the diluent 1;

sample processing

Standard processing

Taking 10 mul, 20 mul, 50 mul, 100 mul, 200 mul, 500 mul of mixed standard solution of DHEA, P5, 17OHP5 and DHT with the concentration of 10ng/ml in 6 1ml volumetric flasks, and diluting the mixed standard solution to the scale with the diluent 1 to obtain mixed calibration concentration solution of DHEA, P5, 17OHP5 and DHT. Wherein, the calibration concentration solution of DHEA, P5 and 17OHP5 is 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml and the calibration concentration solution of DHT is 0.01ng/ml, 0.02ng/ml, 0.05ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml and 1 ng/ml. Then respectively taking 300 mul of DHEA, P5, 17OHP5 and DHT 7 mixed calibration concentration solutions and a blank serum matrix solution into 8 2ml centrifuge tubes, adding 1ml of acetonitrile solution of mixed internal standard, carrying out vortex oscillation for 1min, carrying out centrifugation for 10min at 13000r/min, transferring all clear liquid to another 2ml centrifuge tube, carrying out blow-drying by nitrogen at 60 ℃, adding 0.3ml of water into a blow-dried centrifuge tube for slightly carrying out vortex oscillation, adding 1ml of methyl tert-butyl ether into the blow-dried centrifuge tube, carrying out vortex oscillation extraction for 10min, transferring an organic phase to another 2ml centrifuge tube, repeating the operation for extraction once again, and combining the two organic phases; blowing the mixture at 60 ℃ by using nitrogen. Adding 100mmol/l ethoxyamine hydrochloride (solvent is 60% methanol water) into the dried centrifuge tube, sealing and reacting in dark water bath for 60 min. After the reaction is finished, centrifuging at 13000r/min for 5min, taking the supernatant, and sampling 10 mul to test on a machine.

Quality control material treatment

300 mul of quality control material with low, medium and high concentrations are respectively put into a 2ml centrifuge tube, and then the method is consistent with the standard processing method, which is not described again.

Assay kit composition

The components of the assay kit are shown in Table 3

TABLE 3 assay kit composition

Method verification

1. The method has the following specificity: and (3) processing and performing on-machine analysis by using a blank saliva matrix as a blank sample, and finding that DHEA, DHT, P5, 17OHP5 and corresponding internal standard peak-out time in a spectrogram of the blank sample have no obvious interference.

2. Calibration curve: and (3) establishing a standard curve by adopting an isotope internal standard quantitative method and utilizing Analyst software to calculate the contents of DHEA, DHT, P5 and 17OHP5 in the sample to be detected by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. Linear equations obtained by fitting DHEA, P5 and 17OHP5 in the range of 0.1-10ng/ml and DHT in the range of 0.01-1ng/ml are good in linearity, the linear correlation coefficient r is larger than 0.999, quantitative requirements are met, and results are detailed in table 4.

3. Method minimum detection Limit (LOD) and minimum quantitation Limit (LOQ): the single-standard solutions DHEA, DHT, P5 and 17OHP5 were serially diluted and treated and then repeatedly measured 10 times, so that the S/N ratio at the LOD concentration was 3 or more and the S/N ratio at the LOQ concentration was 10 or more, and the results are shown in Table 4.

And (3) precision test: taking 3 mixed standard solutions with low, medium and high concentrations, wherein each concentration is 3 in parallel, detecting the concentration of 4 steroid hormones after pretreatment, and the precision range in the day is as follows: 1.41 to 8.53 percent. Taking 3 mixed standard solutions with low, medium and high concentrations, each concentration being 9 parallels, processing and detecting the concentration of 4 steroid hormones (each concentration being 3 parallels each day) continuously for 3 days, wherein the precision range between days is as follows: 0.73% -8.53%, and the results are detailed in Table 4.

TABLE 44 Linear equation of steroid hormones, minimum detection limit, minimum quantitation limit and precision test

5. Recovery rate test: selecting a blood sample, dividing into 4 parts, each 300 μ l, wherein one part is not labeled, adding low, medium and high concentration standard substances into the other 3 parts, and processing 4 parts of blood and measuring on a machine for 3 times. The calculated normalized recovery was between 84.0% and 113.3%, and the results are shown in Table 5.

TABLE 54 steroid hormone recovery normalized to ng/ml

Taking 10 mul of mother liquor of DHEA, P5, 17OHP5 and DHT standard products in a 10ml volumetric flask, diluting the mother liquor to a certain volume with a diluent to obtain a mixed standard solution of 1 mug/ml DHEA, P5, 17OHP5 and 0.1 mug/ml DHT; taking 100 mul of the mixed standard solution, and diluting to 10ml with diluent 1 to obtain mixed standard solution of 10ng/ml DHEA, P5, 17OHP5 and 1ng/ml DHT;

respectively taking 10 mul, 20 mul, 50 mul, 100 mul, 200 mul and 500 mul of DHT mixed standard solution with the concentration of 10ng/ml DHEA, P5, 17OHP5 and 1ng/ml DHT to 6 volumetric flasks with 1ml, and diluting the volume to the scale with diluent to obtain DHEA, P5, 17OHP5 and DHT mixed calibration concentration solution; the calibration concentration solutions of DHEA, P5 and 17OHP5 are 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml and 10ng/ml, and the calibration concentration solutions of DHT are 0.01ng/ml, 0.02ng/ml, 0.05ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml and 1 ng/ml;

respectively taking 300 mu l of DHEA, P5, 17OHP5 and DHT 7 mixed calibration concentration solutions and a blank serum matrix solution into 8 2ml centrifuge tubes, adding 1ml of acetonitrile solution of mixed internal standard, carrying out vortex oscillation for 1min, carrying out centrifugation for 10min at 13000r/min, transferring all clear liquid to another 2ml centrifuge tube, carrying out blow-drying by nitrogen at 60 ℃, adding 0.3ml of water into a blow-dried centrifuge tube, slightly carrying out vortex oscillation, adding 1ml of methyl tert-butyl ether, carrying out vortex oscillation extraction for 10min, transferring an organic phase to another 2ml centrifuge tube, repeating the operation for extraction once again, and combining the two organic phases; blowing the mixture at 60 ℃ by using nitrogen. Adding 100mmol/l ethoxyamine hydrochloride and 60% methanol water as solvent into the dried centrifuge tube, sealing and reacting in dark water bath for 60min, centrifuging at 13000r/min for 5min, collecting supernatant, and detecting with 10 μ l machine.

Therefore, the compounds generally need to be modified through derivatization reaction, the ionization efficiency of the modified compounds is obviously improved, and the detection sensitivity is greatly improved.

The method for simultaneously detecting 4 steroid hormones (dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-hydroxypregnenolone) with low ionization efficiency in human blood is established by utilizing a high performance liquid chromatography tandem mass spectrometry technology, and the detection sensitivity of the 4 steroid hormones is improved by 20-100 times by using O-ethylhydroxylamine hydrochloride as a derivatization reagent and performing derivatization treatment on a blood sample after protein precipitation and extraction. The method has the advantages of simple sample pretreatment, stable and easily-preserved derivatization reagent, stable and difficultly-degraded derivatization product, no need of further treatment of a system after reaction and the like, and has accurate and rapid detection result and easy identification.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (8)

1. A method for detecting multiple steroid hormones in blood is characterized in that DHEA, DHT, P5 and 17OHP5 in blood after pretreatment are detected by adopting a high performance liquid chromatography-tandem mass spectrometry technology;

s1, separating DHEA, DHT, P5 and 17OHP5 from interferents by using high performance liquid chromatography;

s2, quantifying by using an isotope internal standard method;

and S3, establishing a standard curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of DHEA, DHT, P5 and 17OHP5 in the sample to be detected.

2. The method for detecting multiple steroid hormones in blood as claimed in claim 1, wherein the specific conditions of high performance liquid chromatography are:

mobile phase A: formic acid water with the volume ratio of 0.1 percent;

mobile phase B: acetonitrile with purity of chromatographic purity; the concentration is 100%;

the type of the chromatographic column: waters XBridge BEH C18, 3.5 μm,2.1mm 150 mm;

a gradient elution mode is adopted, and the method is shown in a table 1;

the flow rate was 0.2ml/min, the column temperature was 40 ℃ and the injection volume was 10. mu.l.

TABLE 1 mobile phase gradient elution parameters

3. The method for detecting multiple steroid hormones in blood as claimed in claim 2, wherein the mass spectrometric conditions are:

under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 5500V; the collision gas is 8; the air curtain air is 20 psi; the ion source temperature is 650 ℃; ion source gas stream GS1 was 70 psi; GS2 at 50 psi; target DHEAm/z 332.2 → 253.1, d6-DHEAm/z 338.2 → 253.1; DHTm/z 334.2 → 105.1, d3-Tm/z 337.2 → 105.1; p5m/z 360.2 → 300.1, d2,¹³C2-P5m/z 364.2→304.1；17OHP5m/z 358.2→145.1，d2,¹³C2-17OHP5m/z 362.2→145.1；

the 5 steroid hormones and the internal standard declustering voltage-DP, injection voltage-EP, collision voltage-CE, collision chamber injection voltage-CXP parameters are shown in Table 2.

TABLE 2 Mass Spectrometry parameters of Testosterone and internal standards

4. The method for detecting multiple steroid hormones in blood according to claim 1, wherein the pretreated blood is prepared by the following method: adding 300 mu l of blood into a 2ml centrifuge tube, adding the mixed internal standard solution, carrying out vortex oscillation for 1min, centrifuging for 10min at 13000r/min, transferring all clear liquid to another 2ml centrifuge tube, carrying out nitrogen blow-drying, adding water into the blow-dried centrifuge tube, slightly carrying out vortex oscillation, adding the extract liquid, carrying out vortex oscillation extraction, transferring an organic phase to another 2ml centrifuge tube, repeating the operation, carrying out extraction once again, and combining the two organic phases; drying by nitrogen;

adding a derivatization agent into the dried centrifugal tube, and sealing and avoiding light for water bath reaction; after the reaction is finished, centrifuging at 13000r/min for 5min, taking the supernatant, and sampling 10 mul to test on a machine.

5. The method for detecting multiple steroid hormones in blood as claimed in claim 4, wherein the internal standard solution is mixed to prepare: accurately weighing 1mg of d6-DHEA, d3-DHT and d2 respectively,¹³C2-P5、d2,¹³dissolving C2-17OHP5 standard substance in 4 10ml volumetric flasks, dissolving with acetonitrile to constant volume to obtain d6-DHEA, d3-DHT and d2 with the concentration of 100 mu g/ml,¹³C2-P5、d2,¹³c2-17OHP5 internal standard mother liquor;

taking 10 mul of d6-DHEA and d2 with the concentration of 100 mug/ml,¹³C2-P5、d2,¹³c2-17OHP5 and 1 mul 100 mug/ml d3-DHT internal standard mother liquor are put into a 10ml volumetric flask, diluted by acetonitrile to be calibrated to obtain 100ng/ml d6-DHEA and d2,¹³C2-P5、d2,¹³c2-17OHP5 and 10ng/ml d3-DHT mixed solution;

diluting 0.25ml of the mixed solution with acetonitrile to 100ml to obtain d6-DHEA and d2 with the concentration of 0.25ng/ml,¹³C2-P5、d2,¹³c2-17OHP5 and 0.025ng/ml d3-DHT mixed internal standard solution for experiments.

6. The method according to claim 4, wherein the volume of the mixed internal standard solution is 1ml, the temperature for blowing dry with nitrogen is 60 ℃, the volume of water added into the blow-dry centrifuge tube is 0.3ml, the extract is methyl tert-butyl ether, the volume of extract added is 1ml, the vortex oscillation extraction time is 10min, the ethoxyamine hydrochloride with the derivatization agent concentration of 100mmol/l is added, the solvent is 60% methanol water, and the volume of the derivatization agent added is 100 μ l; the reaction temperature of the lucifugal water bath is 60 ℃, and the reaction time is 60 min.

7. A kit, comprising the following reagents:

a mobile phase comprising mobile phase A and mobile phase B;

mobile phase A: 0.1% by volume of formic acid aqueous solution;

mobile phase B: acetonitrile, chromatographic purity;

and (3) standard mother liquor: a solution of dehydroepiandrosterone, dihydrotestosterone, pregnenolone, 17-hydroxypregnenolone in acetonitrile;

internal standard solution: d 6-dehydroepiandrosterone, d 3-testosterone, d2,¹³c2-pregnenolone, d2,¹³acetonitrile solution of C2-17-hydroxypregnanolone;

diluting liquid: comprises a first diluent and a second diluent;

diluting a first solution: a blank serum matrix solution, which is prepared by dissolving 1g of bovine serum albumin with water and fixing the volume to 100 ml;

and (2) diluting a second solution: acetonitrile;

extracting liquid: methyl tert-butyl ether;

quality control product: the blank serum matrix solution of dehydroepiandrosterone, pregnenolone and 17-hydroxypregnenolone is divided into low QCL, medium QCM and high QCH concentrations, and the concentrations are respectively as follows: 0.3ng/ml, 3ng/ml and 8 ng/ml; the blank serum matrix solution of dihydrotestosterone is divided into low QCL, medium QCM and high QCH concentrations, and the concentrations are respectively as follows: 0.03ng/ml, 0.3ng/ml and 0.8 ng/ml;

respectively taking 10 mu l of dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-hydroxypregnenolone standard mother liquor in a 10ml volumetric flask, diluting the solution to a certain volume by using a diluent to obtain a mixed standard solution of 1 mu g/ml dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone and 0.1 mu g/ml dihydrotestosterone;

preparing blank serum matrix solution of dehydroepiandrosterone, dihydrotestosterone, pregnenolone and 17-hydroxypregnenolone:

low QCL: taking 3 mul of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

medium QCM: taking 30 mu l of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

high QCH: taking 80 mul of the mixed standard solution, and diluting the mixed standard solution to a constant volume of 10ml by using a diluent;

redissolving the solvent: 65% acetonitrile water solution by volume.

8. A kit according to claim 7, wherein;

preparing a standard mother solution: respectively and accurately weighing 10mg of dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone standard substance and 1mg of dihydrotestosterone standard substance in 4 10ml volumetric flasks, dissolving with acetonitrile and fixing the volume to a scale to obtain 1mg/ml of dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone standard substance mother liquor and 0.1mg/ml of dihydrotestosterone standard substance mother liquor;

preparing an internal standard solution: accurately weighing 1mg of d 6-dehydroepiandrosterone, d 3-testosterone and d2 respectively,¹³c2-pregnenolone, d2,¹³dissolving C2-17-hydroxypregnenolone standard in 4 10ml volumetric flasks, dissolving with acetonitrile, metering to desired volume to obtain d 6-dehydroepiandrosterone, d 3-testosterone, and d2 at a concentration of 100 μ g/ml,¹³c2-pregnenolone, d2,¹³c2-17-hydroxypregnanolone internal standard mother liquor.

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