Method and kit for determining various steroid hormones in body fluid by liquid chromatography-mass spectrometry

Abstract

The invention provides a method and a kit for determining various steroid hormones in body fluid by liquid chromatography-mass spectrometry. The method comprises the following steps: pre-treating a biological fluid sample by introducing an isotope internal standard, a salting-out auxiliary reagent, an extracting agent and an automatic pipetting platform, then separating an isomeride and an interferent through liquid chromatography, switching quantitative ions and qualitative ions with positive-negative scanning characteristics by tandem mass spectrometry, making a standard curve by using a calibrator for quantification, and finally determining the content of the biological fluid sample. And low, medium and high quality control products continuously monitor the performance of the method. According to the method, the aim of simultaneously analyzing and detecting 28 steroid hormones and 24 D / 13C / 15N labeled isotope hormones in a sample by using a UPLC-MS / MS technology without using solid-phase extraction and non-derivatization treatment is achieved, and the method is rapid, simple and convenient to operate, multiple in detection variety, high in flux, high in specificity, low in cost, high in sensitivity and high in sensitivity. 28 indexes of a steroid hormone metabolic pathway are effectively and systematically monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of steroid hormone detection, and specifically relates to a method and a kit for determining multiple steroid hormones in body fluids by using liquid chromatography-mass spectrometry technology. Background Art

[0002] Steroid hormones are a class of small molecule steroid hormones with a cyclopentane polyhydrophenanthrene structure. According to their physiological functions, they can be divided into progesterone, mineralocorticoids, glucocorticoids, estrogens and androgens. Steroid hormones play an important regulatory role in human metabolism, growth, development, reproductive behavior, and maintaining internal environment stability. Clinically, steroid hormone levels are used as diagnostic indicators for many diseases, including congenital steroid metabolic disorders (such as congenital adrenal hyperplasia, congenital developmental disorders, and congenital salt homeostasis disorders) and acquired steroid metabolic disorders (such as primary aldosteronism, polycystic ovary syndrome, Cushing's syndrome, Addison's disease, hyperandrogenism, and mental status disorders). In addition, there are some exogenous steroid hormones, such as dexamethasone and prednisolone, which are mainly artificially synthesized steroid hormone drugs used to treat related diseases.

[0003] Steroid hormones have low content in the body, similar structures, and a wide variety of types, so accurate quantification of steroid hormones has high requirements for the sensitivity of the instrument and the accuracy of the method. At present, the main clinical detection methods for steroid hormones are chemical immunoassay, LC-MS / MS, and gas chromatography-mass spectrometry. Chemical immunoassay is widely used in clinical practice because of its simple and rapid operation. However, traditional chemical immunoassay has many defects and has gradually been replaced by chromatography. Gas chromatography-mass spectrometry has high sensitivity, but often requires cumbersome derivatization steps and long analysis time (generally ≥30min), and there is currently a lack of a derivatization reagent suitable for all steroid hormones. LC-MS / MS technology avoids the cumbersome derivatization steps of gas chromatography, has strong specificity, and can well eliminate the interference of similar structural substances in the matrix. It is currently the most reliable detection method. In the LC-MS / MS detection method, there are also pre-treatment methods through chemical derivatization or solid phase extraction, but it will increase the complexity of the operation and high cost.

[0004] In addition, the indicators of steroid hormone metabolic pathways currently covered by LC-MS / MS technology for steroid hormone detection are not comprehensive enough, and cannot better help researchers accurately detect the concentration of each steroid hormone and understand their metabolic pathways. Summary of the invention

[0005] In response to the problems in the detection of steroid hormones in the prior art, the first object of the present invention is to provide a method for determining multiple steroid hormones in body fluids using liquid chromatography-mass spectrometry. The method is fast and easy to operate, has a wide range of detection types, high throughput, high specificity, and low cost, and can effectively and systematically monitor 28 indicators of the full pathway metabolism of steroid hormones.

[0006] The second object of the present invention is to provide a kit for determining multiple steroid hormones in body fluids using liquid chromatography-mass spectrometry technology. The kit not only improves the efficiency and accuracy of detection, but also reduces the detection cost, simplifies the operation process, and provides a powerful tool for the scientific research of endocrine diseases.

[0007] In order to achieve the above technical purpose, the present invention provides a method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry, wherein the method comprises adding a body fluid sample containing steroid hormones, an isotope internal standard, a salting-out auxiliary reagent and an extractant to a 96-well plate, mixing and extracting, and concentrating the extract to obtain a sample to be tested; the sample to be tested is detected by UPLC-MS / MS;

[0008] The salting-out auxiliary reagent is at least one of saturated sodium bicarbonate and ammonium carbonate solution;

[0009] The extractant is at least one of methyl tert-butyl ether, n-hexane, ethyl acetate and chloroform. More preferably, the extractant is methyl tert-butyl ether, ethyl acetate and n-hexane in a volume ratio of (1-3): (1-2): 1.

[0010] The technical solution of the present invention does not require solid phase extraction and derivatization treatment. The key lies in the use of salting-out auxiliary reagents and extractants in the pre-treatment process. The salting-out auxiliary reagent can change the ionic strength of the sample solution by increasing the salt concentration, thereby promoting the transfer of the target analyte from the aqueous phase to the organic phase, thereby increasing the distribution coefficient of the target analyte in the organic phase. The extract can be further purified by liquid-liquid extraction to remove other components not affected by the salting-out effect, thereby improving the purity of the final analyte. Through the pre-treatment process of the present invention, 28 steroid hormones and 24 isotope-labeled internal standards can be effectively extracted, and combined with UPLC-MS / MS detection, rapid and accurate detection of 28 steroid hormones in body fluids can be achieved.

[0011] The inventors found that the addition of a salting-out auxiliary agent in the present invention can also assist in improving the response value of estrogen and improving the appearance of miscellaneous peaks under the chromatographic conditions of the present invention. It is further preferred that the amount of the salting-out auxiliary agent is 10 to 50 μl of 100 mg / ml NaHCO 3 .

[0012] As a preferred embodiment, the body fluid sample includes serum, plasma, urine, amniotic fluid and cerebrospinal fluid.

[0013] As a preferred embodiment, the steroid hormone includes at least two of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, estrone, estradiol, estriol, dexamethasone, 18-hydroxycorticosterone, 18-oxycortisol, 18-hydroxycortisol, 11β-hydroxytestosterone, 11β-ketotestosterone, androsterone, prednisolone and epitestosterone.

[0014] As a preferred embodiment, the isotope internal standard includes at least two of D4-pregnenolone, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-[d5], D4-cortisol, cortisone-[D7], dehydroepiandrosterone-[D6], dehydroepiandrosterone-[D6] sulfate sodium salt, androstenedione-d7, D4-testosterone, dihydrotestosterone-D4, D4-estrone, estradiol-D4, estriol-D3, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, 11β-ketotesterone-d3 and androsterone-[d4].

[0015] As a preferred solution, the UPLC-MS / MS conditions are:

[0016] Chromatographic conditions:

[0017] Chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; column temperature: 40 ° C; eluent A: methanol-water solution containing ammonium fluoride; eluent B: pure methanol solution, flow rate 0.35 mL / min, injection volume, 10 μL; gradient elution: 0-0.5 min, 0% B; 0.5-0.6 min, 0-28.5% B; 0.6-5.1 min, 28.5-30% B; 5.1-5.2 min, 30-3 9%B; 5.2~7.4min, 39~40%B; 7.4~7.5min, 40~60%B; 7.5~8.9min, 60%B; 8.9~9.5min, 60~85%B; 9.5~9.6min, 85~100%B; 9.6~10.2min, 100%B; 10.2~10.25min, 100~0%B; 10.25~11.5min, 0%B;

[0018] Mass spectrometry conditions: in electrospray ionization (ESI) positive and negative ion switching mode; using multiple reaction monitoring (MRM) mass spectrometry scanning mode, capillary voltage (Capillary): 1.5 kV; desolvation gas temperature: 650 ° C; desolvation gas flow rate: 800 L / Hr; simultaneous monitoring of the target and isotope internal standard.

[0019] As a preferred solution, the concentration is performed by removing the supernatant by centrifugation using an automated platform, nitrogen blowing, re-dissolving, and shaking centrifugation.

[0020] As a preferred solution, the automation platform uses a genetic metabolism sample pretreatment system, an air replacement pipetting mode, and a 4-channel automated pipetting device to transfer the supernatant from the extraction plate to the upper machine plate. Further preferably, the automation platform is the genetic metabolism sample (liquid chromatography-tandem mass spectrometry) pretreatment system (Zhehang Machinery Preparation No. 20200789) manufactured by Bosheng Intelligent Manufacturing, and the automated transfer plate program is described as follows: start pipetting → the pipetting channel takes the gun tip from the gun tip position → the pipetting channel aspirates at the extraction position → the pipetting channel spits out liquid at the upper machine position → the pipetting channel removes the gun tip in the waste tank → the whole plate pipetting ends.

[0021] As a preferred solution, the reconstitution adopts eluent A and / or eluent B as the reconstitution solution.

[0022] The second object of the present invention is to provide a kit for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry, comprising an eluent, an isotope internal standard solution, a salting-out auxiliary reagent, an extractant, a calibration diluent, a quality control product, a reconstitution solution and a chromatographic column; the eluent comprises an eluent A and an eluent B, wherein the eluent A is an ammonium fluoride methanol aqueous solution, and the eluent B is a pure methanol solution.

[0023] As a preferred solution, the concentration of the eluent A is 0.05-0.5 mmol / L of ammonium fluoride methanol aqueous solution.

[0024] As a preferred solution, the calibration substance dilution solution is prepared by diluting a mixed standard solution of seven concentrations by adding a calibration line matrix into a mixed standard working solution.

[0025] As a preferred solution, the calibration line matrix is ​​a substitute matrix consisting of bovine serum albumin and phosphate buffered saline solution.

[0026] As a preferred solution, the quality control product contains three levels of low, medium and high concentration quality control serum. The quality control product is prepared by adding mixed hormone standard substances to serum blank samples purified by activated carbon or hormone-free serum or hormone-free plasma, and the target value is determined by detection.

[0027] As a preferred solution, the seven concentrations of the mixed standard solution corresponding to the dilution multiples of the mixed standard S7 working solution are 1, 2, 5, 10, 25, 50, and 100 times respectively.

[0028] As a preferred embodiment, in the mixed standard working solution, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, estrone, estradiol, estriol, dexamethasone, 18-hydroxycorticosterone, 18-oxycortisol, 18-hydroxycortisol, 11β-hydroxytestosterone, 11β The concentrations of -ketone testosterone, androstenone, prednisolone, and epitestosterone were 20000, 5000, 250, 5000, 500, 10000, 5000, 200, 1000, 100000, 20000, 7500, 2000000, 2000, 2500, 1250, 500, 1000, 200, 5000, 1000, 200, 1000, 200, 800, 4000, 2500, and 500 ng / dl, respectively.

[0029] As a preferred embodiment, the isotope internal standard solution includes D4-pregnenolone, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-[d5], D4-cortisol, cortisone-[D7], dehydroepiandrosterone-[D6], dehydroepiandrosterone-[D6] sodium sulfate, androstenedione-d7, D4-testosterone, dihydrotestosterone-D4, D4-estrogen The concentrations of estradiol-D4, estriol-D3, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, 11β-ketotetestosterone-d3, and androstenone-[d4] were 4000, 2000, 100, 2000, 200, 4000, 2000, 80, 400, 40000, 8000, 3000, 800000, 800, 1000, 500, 200, 400, 80, 2000, 400, 400, 320, and 1600 ng / dl, respectively.

[0030] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0031] (1) The method provided by the present invention for determining 28 steroid hormones in body fluids by liquid chromatography-mass spectrometry has a quick and simple pretreatment process, and does not require the use of complex solid-phase extraction and derivatization treatment. The 28 steroid hormones and 24 isotope-labeled internal standards can be effectively extracted only through a salting-out assisted liquid-liquid extraction process. Combined with the use of a 96-well plate and an automated pipetting platform, the pretreatment step operation is greatly simplified and the throughput is improved, thus meeting the detection needs of large quantities of samples.

[0032] (2) The detection method of the present invention covers 28 indicators on the steroid hormone metabolic pathway, including progesterone, mineralocorticoids, glucocorticoids, estrogens, androgens and exogenous hormones, covering a wide range and meeting the requirements for the detection of multiple steroid hormones in one experiment.

[0033] (3) The detection method of the present invention has high specificity and high accuracy. First, by optimizing the chromatographic conditions, the seven groups of isomers and other interfering substances are baseline separated, effectively avoiding the influence of isomers and other interfering substances on the index determination results; secondly, the treated analyte is directly detected according to the inherent mass / charge ratio, rather than the traditional indirect detection of derivatives, and the method has strong specificity; finally, 24 D / 13C / 15N labeled isotopes are introduced as internal standard quantification, which reduces the influence of the matrix effect between different matrices on the quantitative results.

[0034] (4) The kit provided by the present invention uses calibrators to prepare standard curve quantification, and three levels of quality control products, low, medium and high, continuously monitor the performance of the method, which can more conveniently provide stable and reliable detection of steroid hormones in the entire metabolic pathway for non-medical treatment purposes.

[0035] (5) Add NH 4 F, which is beneficial to improving the negative ion substance response of the steroid hormone measured by the present invention and improving the peak shape and separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the LC-MS / MS ion chromatogram of the simultaneous detection of 28 hormones in the steroid hormone metabolic pathway in Example 1.

[0037] Figure 2 Figure 2 shows the ion currents of isomers Aldo, CT and PNSL.

[0038] Figure 3 Figure 2 is the ion flow diagram of isomers 18OH-CORT and COR.

[0039] Figure 4 The ion current diagrams of isomers 21-DOC, 11-DOC and CORT.

[0040] Figure 5Ion chromatograms of isomers T, DHEA and EPI-T.

[0041] Figure 6 The ion current diagram of isomers 17α-OHP4 and 21-OHP.

[0042] Figure 7 Figure 2 shows the ion chromatogram of isomers ANDR and interfering substances.

[0043] Figure 8 Figure 2 shows the ion chromatogram of isomer P5 and interferences.

[0044] Fig. 9 This is a standard curve diagram of 17α-OHP4 established based on the standard substance according to the example of the present invention.

[0045] Fig.10 This is a standard curve diagram of T based on standard substances in an embodiment of the present invention.

[0046] Fig.11 1 is a comparative ion flow diagram of a normal person and a sample with abnormal progesterone metabolism according to an embodiment of the present invention.

[0047] Fig.12 This is a comparative ion flow diagram of a normal T person and a sample with abnormal androgen metabolism according to an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0050] The method and kit of the present invention are further described by the following examples in conjunction with the accompanying drawings, but these examples do not constitute any limitation to the present invention.

[0051] In the method of the present invention, a 96-well plate is used in combination with an automated pipetting platform. The automated pipetting platform uses the genetic metabolism sample (liquid chromatography-tandem mass spectrometry) pretreatment system (Zhehang Machinery No. 20200789) manufactured by Bosheng Intelligent Manufacturing. The automated plate transfer program description: using an air displacement pipetting mode, the 4-channel automated pipetting device transfers the supernatant from the extraction plate to the upper plate, automatically replaces the gun tip, and some program parameter settings are as follows:

[0052] Liquid aspiration position: the center of the plate hole is 10 mm away from the bottom of the plate hole;

[0053] Aspiration volume setting: 350 μl;

[0054] Aspiration speed: 80 μl / s;

[0055] Aspirate air after aspirating liquid: 5 μl;

[0056] The discharge volume setting: 350 μl;

[0057] Dispensing position: The center of the plate hole is 5 mm away from the bottom of the plate hole;

[0058] Dispensing setting: Liquid level following mode (when dispensing, the pipette rises with the liquid level);

[0059] Dispensing speed: 200μl / s.

[0060] The abbreviations of various steroid hormones involved in the present invention are shown in Table 1 below.

[0061] Table 1 Abbreviations of steroid hormones

[0062]

[0063]

[0064] Example 1

[0065] This embodiment is a method and kit for determining multiple steroid hormones in body fluids using liquid chromatography-mass spectrometry, as follows:

[0066] A method for determining multiple steroid hormones in body fluids using liquid chromatography-mass spectrometry.

[0067] 1. Preparation of Calibrators

[0068] The dissolution of the standards and the preparation of concentrated stock solutions are shown in Table 2.

[0069] Mix the concentrated stock solutions in Table 2 by pipetting and add 9331 μL of 80% methanol to obtain the mixed standard working solution.

[0070] The steroid hormone calibrators were prepared by adding seven concentrations of mixed standard solutions to the mixed standard working solution for dilution, and the calibration line matrix was a surrogate matrix consisting of bovine serum albumin and phosphate buffered saline.

[0071] The seven concentration points S1-S7 of the calibrator were obtained by serially diluting the high-concentration calibrator with the calibration line matrix. The dilution multiples were 2000, 1000, 500, 200, 100, 40, and 20, respectively. The concentrations of the calibration points S1-S7 are shown in Table 3.

[0072] Table 2 Concentrations of steroid hormone stock solutions and mixed standards

[0073]

[0074]

[0075] Table 3 Concentrations of various steroid hormones at different concentration levels of calibration points S1-S7

[0076]

[0077]

[0078] 2. Preparation of quality control products

[0079] Quality control spike concentration setting and preparation process: The quality control products contain three levels of low, medium and high concentration quality control serum. The quality control products are prepared by adding mixed hormone standard substances to serum blank samples purified by activated carbon or hormone-free serum / plasma. The concentrated stock solution is mixed with the serum blank sample and diluted 500, 200 and 40 times respectively to obtain three levels of low, medium and high quality control products. The low, medium and high quality control products are represented by L, M and H respectively. The concentrations of steroid hormones are shown in Table 4.

[0080] Table 4 Concentration of steroid hormones in quality control products

[0081]

[0082]

[0083] 3. Liquid chromatography and mass spectrometry conditions

[0084] Chromatographic conditions:

[0085] Eluent A: 10 vol% methanol + 90 vol% water + 0.05 mM ammonium fluoride;

[0086] Eluent B: methanol;

[0087] Chromatographic column: ACQUITY UPLC BEH C18 column, 50 mm × 2.1 mm, 1.7 μm; flow rate: 0.35 mL / min; column temperature: 40 °C; injection volume: 10 μL; gradient elution was performed, and the gradient elution program is shown in Table 5.

[0088] Table 5 Gradient elution program

[0089]

[0090] Mass spectrometry conditions:

[0091] In the electrospray ionization (ESI) positive and negative ion switching mode; using the mass spectrometry scanning mode of multiple reaction monitoring (MRM), the capillary voltage (Capillary): 1.5kV; the desolvation gas temperature: 650℃; the desolvation gas flow rate: 800L / Hr; the target object and the isotope internal standard were monitored simultaneously.

[0092] The mass spectrometry parameters for the detection of 28 steroid hormones in multiple reaction monitoring mode are shown in Table 6.

[0093] The mass spectrometry parameters for detecting 24 isotope-labeled steroid hormones in multiple reaction monitoring mode are shown in Table 7.

[0094] Table 6 Mass spectrometry parameters of steroid hormones

[0095]

[0096]

[0097] Note: * in the table indicates (quantitative ion pair).

[0098] Table 7 Mass spectrometry parameters of isotope-labeled steroid hormones

[0099]

[0100]

[0101] Note: * in the table indicates quantitative ion pairs.

[0102] 4. Experimental process

[0103] Preparation of isotope internal standard solution:

[0104] Dissolve and prepare the internal standard mixed solution of the internal standard according to the preparation steps of the standard solution, D4-pregnenolone, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2;2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-[d5], D4-cortisol, cortisone-[D7], dehydroepiandrosterone-[D6], dehydroepiandrosterone-[D6] sulfate sodium salt, androstenedione-d7, D4-testosterone, dihydrotestosterone-D4, The concentrations of D4-estrone, estradiol-D4, estriol-D3, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, 11β-ketotetestosterone-d3, and androstenone-[d4] were 4000, 2000, 100, 2000, 200, 4000, 2000, 80, 400, 40000, 8000, 3000, 800000, 800, 1000, 500, 200, 400, 80, 2000, 400, 400, 320, and 1600 ng / dl, respectively.

[0105] Preparation of salting-out auxiliary reagents:

[0106] Accurately weigh 100 mg of sodium bicarbonate and dissolve it in 1 ml of pure water to prepare a 100 mg / ml saline solution.

[0107] Selection of extraction agent:

[0108] The extractant was a mixed solution of methyl tert-butyl ether (MTBE), ethyl acetate (EA) and n-hexane (n-HEX) in a volume ratio of 1:2:1.

[0109] Choice of reconstitution solution:

[0110] The reconstitution solution is a solution in which the volume ratio of eluent A to eluent B is 1:0.8.

[0111] Sample processing:

[0112] 1) Take 200 μl of plasma into a 1 ml 96-well plate, add 20 μl of isotope internal standard working solution and 20 μl of 100 mg / ml (saturated) sodium bicarbonate solution in sequence, cover with a 96-well silicone cover plate and mix thoroughly;

[0113] 2) Remove the 96-well silica gel cover, pipette 500 μL of the extractant MTBE:EA:n-HEX=1:2:1) into the 96-well plate, cover the 96-well silica gel cover with a clean 96-well silica gel cover, vortex thoroughly, replace with a clean 96-well silica gel cover, and centrifuge at 3000 r / min and 10°C for 10 min;

[0114] 3) Turn on the pipetting platform and software, wait for the device self-check to end, open the pipetting platform door, pull out the sample processing rack (pipetting module), place a clean 400μl 96-well plate, a 1ml 96-well plate to be pipetted, and a sufficient amount of 1ml pipetting platform gun tips on the upper plate, extraction plate, and gun tip positions respectively, return the sample processing rack (pipetting module) to its original position, close the door, and click Start on the software.

[0115] 4) After the pipetting platform operation is completed, place the 400 μl 96-well plate under a gentle nitrogen flow to remove the solvent, discard the 1 ml 96-well plate and the disposable gun holder of the gun tip, and turn off the software and the power of the pipetting platform.

[0116] 5) Redissolve the residue in 40 μl of reconstitution solution and load the sample after it is fully dissolved. The injection volume is 10 μl.

[0117] The LC-MS / MS ion chromatograms of 28 hormones in the steroid hormone metabolic pathway were detected and shown in Figure 1 , the ion flow diagrams of each compound and their isomers are shown in Figure 2 to Figure 8 .

[0118] The method of this embodiment is used to detect samples with abnormal progesterone metabolism, samples with abnormal androgen metabolism and samples from normal people to obtain comparative ion current graphs. Fig.11 and Fig.12 It can be seen from the figure that 17a-OHP4 and P4, which are abnormal progesterone metabolism, and T and DHT, which are abnormal androgen metabolism, are significantly increased.

[0119] Method Validation:

[0120] After the sample to be tested enters the chromatographic column for separation in the UPLC gradient elution mode, the multiple ion reaction monitoring mode (MRM) of positive / negative ion electrospray ionization (ESI) is used to perform qualitative and / or quantitative MS / MS detection on the substance to be tested. A standard curve is prepared using a mixed standard solution of seven concentrations, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, and the peak area of ​​the corresponding substance to be tested is substituted into the regression equation to calculate the concentration of the substance to be tested in the sample; the measured linear results are shown in Table 8. The standard curve diagram for substances 17α-OHP4 and T based on the standard is shown in Fig. 9 and Fig.10 .

[0121] Table 8 Linearity results of tested substances

[0122]

[0123]

[0124] The linear ranges of the 28 detection indicators in this embodiment are all greater than or equal to 2 orders of magnitude, with a wide linear range, meeting the accurate quantitative requirements of samples of different concentrations. The 7 calibration points set, the linear equations of each indicator r 2 The values ​​are all ≥0.99, with excellent linear performance.

[0125] The present invention prepared three quality control samples of L, M, and H by adding known concentrations of standard samples to serum blank samples, and then tested each concentration 6 times. The theoretical value is the sum of the concentration of endogenous analytes in the sample and the concentration of the added standard sample. The ratio of the measured value to the theoretical value can be used to evaluate the accuracy. The range of spiked recovery of specific substances is shown in Table 9.

[0126] Table 9 Sample spike recovery results

[0127]

[0128]

[0129] The detection value of the present invention is within ±15% of the target value, and the method has good accuracy.

[0130] Using the method of the present invention, the detection limits and quantitative limits of 28 detection indicators are evaluated together with the precision and correctness. At least 3 analysis batches are inspected for at least two days, and 6 parallel tests are performed on each sample. The RE and coefficient of variation (CV) are used to determine that the RE is within ±20% and the CV is ≤20%. The detection limits and quantitative limits are shown in Table 10.

[0131] Using the method of the present invention, three different concentrations of quality control products were selected: low, medium and high. To evaluate the intra-batch precision, each concentration was measured 6 times. To evaluate the inter-batch precision, three analytical batches should be prepared and measured continuously in different batches. The specific results are shown in Table 10. The requirements that the low value CV near the LLMI concentration should be ≤20% and the CV of other concentrations should be ≤15% were met.

[0132] Table 10

[0133]

[0134]

[0135] Six batches of blank matrices of different individuals were investigated. By comparing the signals of the analytes spiked into the extracted samples with the signals of the analytes spiked into the standard curve matrix after extraction, the matrix factors of each analyte (low, medium and high quality control concentrations) and internal standard were calculated by calculating the ratio of the peak area response in the presence of matrix (measured by adding analytes and internal standards after extraction of the blank matrix) to the corresponding peak area response without matrix (pure solutions of analytes and internal standards). The matrix factors normalized by the internal standard were calculated. The results are shown in Table 11.

[0136] Table 11

[0137]

[0138]

[0139]

[0140] As shown in the table above, the coefficient of variation (CV) of the matrix factor (ME) of 6 different random blood matrix samples is ≤15%, and the RE of the matrix factor ME% normalized by the internal standard is within ±20%. The introduction of the corresponding 24 D / N / 13C labeled isotopes as internal standard quantification reduces the influence of the matrix effect between different matrices on the quantitative results. The matrix effect is manifested as no obvious inhibition or enhancement, and has quantitative consistency.

[0141] The present embodiment provides a kit for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry, which is composed of an eluent, an isotope internal standard solution, a salting-out auxiliary reagent, an extractant, a calibration diluent, a low-level quality control serum, a mid-level quality control serum, a high-level quality control serum, a reconstitution solution and a chromatographic column; the eluent includes an eluent A and an eluent B, wherein the eluent A is an aqueous solution of ammonium fluoride in methanol, and the eluent B is a pure methanol solution.

[0142] Example 2: Optimization of eluent additives and buffer salt concentration

[0143] The introduction of alkaline additives will enhance the ionization efficiency of negative ion substances. Under other conditions unchanged, the eluent A is compared with the NH 4 F methanol aqueous solution and alkaline additive NH 3 H 2 O methanol aqueous solution. The results show that the positive ion response is not much different, and the addition of NH 4 The overall response of F anions increased, especially E1, E3, and 17α-OHP5. 4 When the molar concentration of F is within the range of 0.05-0.5 mM, the positive ion response does not change significantly, and the effect on the peak time and separation degree does not change significantly. 4 When the molar concentration of F is 0.05 mM, the negative ion substance has the highest response. Therefore, in the method of the present invention, 0.05 mM concentration of NH 4 F. In eluent B, with the same sample and the same injection method, NH 4 F and pure methanol had no significant effect on the elution time and no significant difference in separation degree. Adding methanol to phase B had no improvement on the separation degree, negative ion index and DHEA / P5. Therefore, a pure methanol system can be selected as eluent B.

[0144] Example 3: Selection and optimization of salting-out auxiliary reagents

[0145] Compare whether or not to add salting-out auxiliary reagent after salting-out treatment: The results show that when salting-out auxiliary reagent is added, the response of estrogen E1, E2, P4, 17α-OHP4, 17α-OHP5, 11-DOC, DHEA, DXMS, 18OH-CORT and other peaks will increase, and the peaks of 21-OHP and 17α-OHP5 will be improved, so salting-out extraction is chosen.

[0146] Choice of salt type: When other conditions are the same, equal amounts of NaCl, NaOAc, and NH 4 FA showed no significant improvement compared with no salt addition. 4 ) 2 CO 3 and NaHCO 3 Basically the same, but NaHCO 3 The DHEAS peak shape is relatively better.

[0147] Selection of salt amount: The DHEAS peak shape can be obtained at 10-50 μl. However, as the amount of salt increases, the DHEAS peak moves forward and the sample becomes more turbid. Therefore, in the present invention, 20 μl of 100 mg / ml (saturated) NaHCO is added to each sample. 3 .

[0148] Example 4: Selection and optimization of extraction agent

[0149] Comparison of different extractants - methyl tert-butyl ether (MTBE), n-hexane (n-HEX), ethyl acetate (EA), chloroform (TCM): The extractant of pure n-HEX increased, and the response of multiple indicators including estrogen was low. Pure MTBE 17α-OHP5 has an interference peak, and the response of DHEA, 21-OHP, etc. is low. The extractant containing TCM will be in the lower layer of the sample after high-speed centrifugation, which increases the difficulty of operation to a certain extent. Mixtures of methyl tert-butyl ether (MTBE), n-hexane (n-HEX), and ethyl acetate (EA) in different volume ratios, MTBE:EA:n-HEX=1:2:1 and MTBE:EA:n-HEX=3:1:1 have relatively high responses. Considering the safety of organic reagents, the optimal ratio selected in the present invention is: MTBE:EA:n-HEX=1:2:1.

[0150] Example 5: Selection and optimization of 96-well plates and automated pipetting platforms

[0151] In the pretreatment process of conventional body fluids, especially in the method with liquid-liquid extraction step, centrifuge tubes are often used for operation. During the pretreatment process, the centrifuge tubes are repeatedly removed, which greatly limits the detection throughput and increases the incidence of erroneous operations. In the method of the present invention, a 96-well plate is used in combination with the use of an automated pipetting platform, which greatly simplifies the pretreatment step operation and improves the throughput, meets the detection needs of large batches of samples, and reduces the pretreatment process of 96 samples before loading to about 1 hour.

[0152] The automated pipetting platform uses the genetic metabolism sample (liquid chromatography-tandem mass spectrometry) pretreatment system (Zhejiang-Hangzhou Machinery No. 20200789) manufactured by Bosheng Intelligent Manufacturing. The automated plate transfer program description: using air displacement pipetting mode, the 4-channel automated pipetting device transfers the supernatant from the extraction plate to the upper plate, automatically replaces the gun tip, and some program parameter settings are as follows:

[0153] Liquid aspiration position: the center of the plate hole is 10 mm away from the bottom of the plate hole;

[0154] Aspiration volume setting: 350 μl;

[0155] Aspiration speed: 80 μl / s;

[0156] Aspirate air after aspirating liquid: 5 μl;

[0157] The discharge volume setting: 350 μl;

[0158] Dispensing position: The center of the plate hole is 5 mm away from the bottom of the plate hole;

[0159] Dispensing setting: Liquid level following mode (when dispensing, the pipette rises with the liquid level);

[0160] Dispensing speed: 200μl / s.

[0161] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for determining multiple steroid hormones in body fluids using liquid chromatography-mass spectrometry, characterized in that: A body fluid sample containing steroid hormones, an isotope internal standard, a salting-out auxiliary reagent and an extractant are added to a 96-well plate, mixed and extracted, and the extract is concentrated to obtain a sample to be tested; the sample to be tested is detected by UPLC-MS / MS; The salting-out auxiliary reagent is at least one of saturated sodium bicarbonate and ammonium carbonate solution; The extractant is at least one of methyl tert-butyl ether, n-hexane, ethyl acetate and chloroform.

2. The method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The body fluid samples include serum, plasma, urine, amniotic fluid and cerebrospinal fluid; The steroid hormones include at least two of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, estrone, estradiol, estriol, dexamethasone, 18-hydroxycorticosterone, 18-oxycortisol, 18-hydroxycortisol, 11β-hydroxytestosterone, 11β-ketotestosterone, androsterone, prednisolone and epitestosterone.

3. The method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 2, characterized in that: The isotopic internal standard includes at least two of D4-pregnenolone, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-[d5], D4-cortisol, cortisone-[D7], dehydroepiandrosterone-[D6], dehydroepiandrosterone-[D6] sulfate sodium salt, androstenedione-d7, D4-testosterone, dihydrotestosterone-D4, D4-estrone, estradiol-D4, estriol-D3, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, 11β-ketotestosterone-d3 and androsterone-[d4].

4. The method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to any one of claims 1 to 3, characterized in that: The conditions of the UPLC-MS / MS are: Chromatographic conditions: Chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; Column temperature: 40°C; eluent A: methanol-water solution containing ammonium fluoride; eluent B: pure methanol solution, flow rate 0.35 mL / min, injection volume, 10 μL; gradient elution: 0-0.5 min, 0% B; 0.5-0.6 min, 0-28.5% B; 0.6-5.1 min, 28.5-30% B; 5.1-5.2 min, 30-39% B; 5.2-7.4 min n, 39~40%B; 7.4~7.5min, 40~60%B; 7.5~8.9min, 60%B; 8.9~9.5min, 60~85%B; 9.5~9.6 min, 85~100%B; 9.6~10.2min, 100%B; 10.2~10.25min, 100~0%B; 10.25~11.5min, 0%B; Mass spectrometry conditions: in electrospray positive and negative ion switching mode; using multiple reaction monitoring mass spectrometry scanning mode, capillary voltage: 1.5 kV; desolvation gas temperature: 650 ° C; desolvation gas flow rate: 800 L / Hr; simultaneous monitoring of target and isotope internal standard.

5. The method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 4, characterized in that: The concentration comprises the following steps: removing the supernatant by centrifugation using an automated platform, nitrogen blowing, re-dissolving, and shaking centrifugation; The reconstitution adopts eluent A and / or eluent B as the reconstitution solution.

6. The method for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 5, characterized in that: The automated platform uses a genetic metabolism sample pre-treatment system, an air displacement pipetting mode, and a 4-channel automated pipetting device to transfer the supernatant from the extraction plate to the upper plate.

7. A kit for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to any one of claims 1 to 6, characterized in that: Including eluent, isotope internal standard solution, salting-out auxiliary reagent, extraction agent, calibration diluent, quality control product, reconstitution solution and chromatographic column; The eluent includes eluent A and eluent B, wherein the eluent A is an aqueous solution of ammonium fluoride in methanol, and the eluent B is a pure methanol solution.

8. The kit for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 7, characterized in that: The calibration dilution solution is prepared by adding a calibration line matrix to a mixed standard working solution to dilute a mixed standard solution of seven concentrations; The calibration line matrix is ​​a substitute matrix consisting of bovine serum albumin and phosphate buffered saline; The seven concentrations of the mixed standard solution corresponding to the dilution multiples of the mixed standard S7 working solution are 1, 2, 5, 10, 25, 50, and 100 times respectively; In the mixed standard working solution, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, estrone, estradiol, estriol, dexamethasone, 18-hydroxycorticosterone, 18-oxycortisol, 18-hydroxycortisol, 11β-hydroxytestosterone, 11β-ketotestosterone, The concentrations of androstenone, prednisolone, and epitestosterone were 20000, 5000, 250, 5000, 500, 10000, 5000, 200, 1000, 100000, 20000, 7500, 2000000, 2000, 2500, 1250, 500, 1000, 200, 5000, 1000, 200, 1000, 200, 800, 4000, 2500, and 500 ng / dl, respectively.

9. The kit for determining multiple steroid hormones in body fluids by liquid chromatography-mass spectrometry according to claim 8, characterized in that: In the isotope internal standard solution, D4-pregnenolone, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-[d5], D4-cortisol, cortisone-[D7], dehydroepiandrosterone-[D6], dehydroepiandrosterone-[D6] sodium sulfate, androstenedione-d7, D4-testosterone, dihydrotestosterone-D4, D4-estrone, estradiol The concentrations of -D4, estriol-D3, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, 11β-ketotetestosterone-d3, and androstenone-[d4] were 4000, 2000, 100, 2000, 200, 4000, 2000, 80, 400, 40000, 8000, 3000, 800000, 800, 1000, 500, 200, 400, 80, 2000, 400, 400, 320, and 1600 ng / dl, respectively.

Images