A kit and method for simultaneously determining multiple steroid hormones in serum

Abstract

The present invention relates to the field of biological detection, and in particular to a kit and method for simultaneously determining multiple steroid hormones in serum. The present invention provides a detection reagent, including: a calibrator, a quality control product, an internal standard, an extractant, a resolvent and a buffer; the extractant includes one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane; the resolvent includes a methanol aqueous solution. The kit provided by the present invention only requires 200 μL of serum sample to achieve the purpose of simultaneously detecting the concentrations of multiple steroid hormones, and only uses a two-step extraction method to accurately determine the content of multiple steroid hormones at extremely low concentrations (ng / mL level) in blood. Compared with the existing technology for detecting multiple steroid hormones, the kit provided by the present invention is simple to operate, sensitive and accurate, has a wide linear dynamic range (400 times), can simultaneously determine multiple steroid hormones, and can be used for routine detection of multiple steroid hormones in clinical serum.

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to a kit and a method for simultaneously determining multiple steroid hormones in serum. Background Art

[0002] Steroid hormones, also known as steroid hormones, are a class of fat-soluble small molecule hormones produced by cholesterol through a series of enzyme-catalyzed metabolism, including mineralocorticoids, glucocorticoids and sex hormones. They are based on cyclopentane polyhydrophenanthrene as the parent nucleus and are divided into androstane, estranes and pregnane according to their chemical structure. Steroid hormones play an important role in maintaining life, regulating the body's material metabolism, promoting the development of sexual organs and maintaining fertility. Steroid hormones are mainly transmitted through the blood and bind to receptors in very small doses to exert their effects, with extremely high specificity. When the level of steroid hormones in the body is disordered, it can cause some diseases, such as congenital steroid metabolism disorders (such as congenital adrenal hyperplasia, congenital developmental disorders and congenital salt homeostasis disorders, etc.) and acquired steroid metabolism disorders (such as primary aldosteronism, Cushing's syndrome, Addison's disease, hyperandrogenism and mental status diseases).

[0003] Steroid hormones are present in very low levels in the body, have similar structures, are of many types, and have large concentration differences, generally in the order of nmol / L and pmol / L, and vary with age and gender. Therefore, accurate quantification of steroid hormones places extremely high demands on the sensitivity of the instrument and the accuracy of the method.

[0004] At present, the detection methods of steroid hormones are mainly chemical immunoassay, gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry. Before the 20th century, chemical immunoassay was widely used in clinical practice because of its simple and rapid operation. However, traditional chemical immunoassay has many defects: 1) low sensitivity. When the sample concentration is very low, a large amount of sample enrichment and purification is required; 2) poor applicability. The antibody of the analyte is easy to cross-react with endogenous substances or metabolites with similar structures, resulting in false positive results; 3) low analysis efficiency. Usually only one substance can be determined at a time, and qualitative and quantitative analysis of multiple hormones cannot be performed simultaneously. Therefore, chemical immunoassay has been gradually 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. Liquid chromatography-tandem mass spectrometry 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.

[0005] Therefore, establishing an analytical method that can accurately measure multiple steroid hormones simultaneously has very important guiding significance for the diagnosis of clinical diseases. Summary of the invention

[0006] In view of this, the present invention provides a kit and method for simultaneously determining multiple steroid hormones in serum. The present application provides a kit for simultaneously determining multiple steroid hormones in serum, including multiple steroid hormone mixed calibration working solutions, multiple steroid hormone mixed quality control working solutions, multiple steroid hormone mixed stable isotope internal standard solutions, a first extractant, a second extractant, a first resolvent, a second resolvent, and a buffer solution. This solution is simple to operate, has high specificity, small batch differences, high sensitivity, and a wide dynamic range, and can meet the clinical detection needs for multiple steroid hormones. The present application also provides a method for simultaneously determining multiple steroid hormones in serum, which does not require derivatization treatment, and extracts and purifies specimens through liquid-liquid extraction, thereby simplifying the detection steps.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a detection reagent, including: a calibrator, a quality control product, an internal standard, an extractant, a resolvent and a buffer;

[0009] The extractant includes one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane;

[0010] The resolvent includes methanol-water solution.

[0011] In some embodiments of the present invention, in the above detection reagent, the volume ratio of the methyl tert-butyl ether to the ethyl acetate is 1:1.

[0012] In some embodiments of the present invention, in the above-mentioned detection reagent, the quality control product includes one or more of cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone or dehydroepiandrosterone sulfate.

[0013] In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of cortisone is 2 and 20 ng / mL; the concentration of cortisol is 5 and 50 ng / mL; the concentration of corticosterone is 0.2 and 2 ng / mL; the concentration of 11-deoxycortisol is 0.1 and 1 ng / mL; the concentration of androstenedione is 0.1 and 1 ng / mL; the concentration of testosterone is 0.15 and 1.5 ng / mL; the concentration of dehydroepiandrosterone is 2 and 20 ng / mL; the concentration of 17-hydroxyprogesterone is 0.2 and 2 ng / mL; the concentration of progesterone is 0.4 and 4 ng / mL; the concentration of 17-hydroxypregnenolone is 1 and 10 ng / mL; and the concentration of dehydroepiandrosterone sulfate is 50 and 500 ng / mL.

[0014] In some embodiments of the present invention, in the above-mentioned detection reagent, the internal standard includes one or more of cortisone-d8, cortisol-d4, corticosterone-d4, 11-deoxycortisol-d7, androstenedione-d7, testosterone-d3, dehydroepiandrosterone-d5, 17-hydroxyprogesterone-d8, progesterone-d9, 17-hydroxypregnenolone-d3 or dehydroepiandrosterone sulfate-d6.

[0015] In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of cortisone-d8 is 15ng / mL; the concentration of cortisol-d4 is 100ng / mL; the concentration of corticosterone-d4 is 1ng / mL; the concentration of 11-deoxycortisol-d7 is 0.5ng / mL; the concentration of androstenedione-d7 is 1ng / mL; the concentration of testosterone-d3 is 0.75ng / mL; the concentration of dehydroepiandrosterone-d5 is 10ng / mL; the concentration of 17-hydroxyprogesterone-d8 is 2.5ng / mL; the concentration of progesterone-d9 is 2ng / mL; the concentration of 17-hydroxypregnenolone-d3 is 5ng / mL; and the concentration of dehydroepiandrosterone sulfate-d6 is 250ng / mL.

[0016] In some embodiments of the present invention, in the above detection reagent, the concentration of the methanol aqueous solution is 30% to 100%.

[0017] In some embodiments of the present invention, in the above detection reagent, the concentration of the methanol aqueous solution is 30%, 50%, 60%, 70%, 80% or 100%.

[0018] In some embodiments of the present invention, in the above-mentioned detection reagent, the calibrator includes one or more of cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone or dehydroepiandrosterone sulfate; the buffer includes one or more of 4-hydroxyethylpiperazineethanesulfonic acid, sodium phosphate or sodium borate buffer.

[0019] In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of cortisone is 0.5-200 ng / mL; the concentration of cortisol is 1.25-500 ng / mL; the concentration of 11-deoxycortisol is 0.025-10 ng / mL; the concentration of androstenedione is 0.025-10 ng / mL; the concentration of testosterone is 0.0375-15 ng / mL; the concentration of dehydroepiandrosterone is 0.5-200 ng / mL; the concentration of 17-hydroxyprogesterone is 0.05-20 ng / mL; the concentration of progesterone is 0.1-40 ng / mL; the concentration of 17-hydroxypregnenolone is 0.25-100 ng / mL; the concentration of hydrogen epiandrosterone sulfate is 12.5-5000 ng / mL.

[0020] In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of cortisone is 200, 50, 10, 5, 1, and 0.5 ng / mL; the concentration of cortisol is 500, 125, 25, 12.5, 2.5, and 1.25 ng / mL; the concentration of 11-deoxycortisol is 10, 2.5, 0.5, 0.25, 0.05, and 0.025 ng / mL; the concentration of androstenedione is 10, 2.5, 0.5, 0.25, 0.05, and 0.025 ng / mL; the concentration of testosterone is 15, 3.75, 0.75, and 0.37 5, 0.075, 0.0375ng / mL; the concentration of the dehydroepiandrosterone is 200, 50, 10, 5, 1, 0.5ng / mL; the concentration of the 17-hydroxyprogesterone is 20, 5, 1, 0.5, 0.1, 0.05ng / mL; the concentration of the progesterone is 40, 10, 2, 1, 0.2, 0.1ng / mL; the concentration of the 17-hydroxypregnenolone is 100, 25, 5, 2.5, 0.5, 0.25ng / mL; the concentration of the hydrogen epiandrosterone sulfate is 5000, 1250, 250, 125, 25, 12.5ng / mL.

[0021] The present invention also provides a detection kit, comprising the above detection reagent and an acceptable auxiliary agent or carrier.

[0022] The present invention also provides the use of the above detection reagent or the above detection kit in preparing a product for detecting steroid hormones.

[0023] In some embodiments of the present invention, in the above application, the steroid hormone includes: one or more of cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone or dehydroepiandrosterone sulfate.

[0024] In some embodiments of the present invention, in the above application, the detection includes taking the detection reagent or the detection kit and mixing it with the sample to be tested, and obtaining the concentration of the analyte in the sample to be tested after quantification.

[0025] In some embodiments of the present invention, in the above application, the mixing comprises the following steps:

[0026] S1: Mix the sample to be tested with the calibrator, the quality control product, and the extractant, centrifuge, and collect the supernatant to obtain a first extraction sample;

[0027] S2: taking the first extraction sample, concentrating it, mixing it with the resolvent, and then mixing it with the extractant, centrifuging it, and collecting the supernatant to obtain a second extraction sample;

[0028] S3: After the second extraction sample is concentrated, it is mixed with the resolvent, centrifuged, and the supernatant is collected to obtain the sample to be tested.

[0029] In some embodiments of the present invention, in the above application, the quantification is performed by high performance liquid chromatography tandem mass spectrometry; the conditions of high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry are: injector temperature is 4°C, column temperature is 50°C, running time is 5min, flow rate is 0.3mL / min, gradient elution;

[0030] The procedure of the gradient elution is as follows:

[0031]

[0032] ; The mobile phase A is a 0-1 mM ammonium fluoride aqueous solution; and the mobile phase B is methanol.

[0033] In some embodiments of the present invention, in the above application, the mass spectrometry conditions in the high performance liquid chromatography tandem mass spectrometry are: the capillary voltage in the positive ion mode is 3.0 kV; the capillary voltage in the negative ion mode is -2.5 kV; the desolvation temperature is 550°C; the desolvation gas flow rate is 800 L / hr; the cone gas flow rate is 150 L / hr; and multiple reaction ion detection.

[0034] In some embodiments of the present invention, in the above application, the mass spectrometry conditions are as follows:

[0035]

[0036]

[0037] In some embodiments of the present invention, in the above application, the quantification adopts the internal standard method; the internal standard method includes taking the concentration of the steroid hormone in the calibrator as the horizontal coordinate (x), measuring the peak area ratio of the steroid hormone to the internal standard in the sample to be tested by the calibrator and the corresponding internal standard, substituting into the linear regression equation, and calculating the concentration of the steroid hormone in the sample to be tested.

[0038] The present invention provides a detection reagent, including: a calibrator, a quality control product, an internal standard, an extractant, a resolvent and a buffer;

[0039] The extractant includes one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane;

[0040] The resolvent includes methanol-water solution.

[0041] The beneficial effects of the present invention include:

[0042] (1) The kit provided by the present invention only requires 200 μL of serum sample to achieve the purpose of simultaneously detecting the concentrations of multiple steroid hormones. Only a two-step extraction method is used to accurately determine the content of multiple steroid hormones at extremely low concentrations (ng / mL level) in the blood. Compared with the existing technology for detecting multiple steroid hormones, the kit provided by the present invention is simple to operate, sensitive and accurate, has a wide linear dynamic range (400 times), and can simultaneously determine multiple steroid hormones. It can be used for routine detection of multiple steroid hormones in clinical serum.

[0043] (2) The kit provided by the present invention is easy to operate, has high specificity, small batch-to-batch variability, high sensitivity, and a wide dynamic range, and can meet the clinical needs for the detection of a variety of steroid hormones.

[0044] (3) The present application provides a method for simultaneously determining multiple steroid hormones in serum without the need for derivatization treatment. The specimens are extracted and purified by liquid-liquid extraction, thereby simplifying the detection steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0046] Figure 1 LC-MS / MS chromatograms of various steroid hormone calibrators are shown;

[0047] Figure 2 Figure 4 shows the LC-MS / MS chromatograms of various steroid hormone calibrators in serum samples. DETAILED DESCRIPTION

[0048] The invention discloses a kit and a method for simultaneously measuring multiple steroid hormones in serum.

[0049] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0050] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0051] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0052] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0053] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0054] The present invention provides a method for simultaneously determining multiple steroid hormones in serum, using a kit, comprising the following steps:

[0055] S1: Add the mixed calibrator working solution and the mixed quality control working solution into the blank matrix to prepare the calibration curve sample and the quality control sample;

[0056] S2: a step of extracting a plurality of steroid hormones in a serum sample, a calibration curve sample and a quality control sample using a first extractant, and taking a supernatant after centrifugation to obtain a first extracted sample;

[0057] S3: After the first extracted sample is dried and concentrated by nitrogen, it is dissolved by a first re-solvent to obtain a first re-dissolved sample;

[0058] S4: extracting impurities in the first reconstituted sample with a second extractant, discarding the supernatant after centrifugation and taking the lower solution to obtain a second extracted sample;

[0059] S5: drying and concentrating the second extracted sample with nitrogen, dissolving it with a second complex solvent, and detecting it with high performance liquid chromatography tandem mass spectrometry;

[0060] The present invention also provides a kit, comprising the following components: a plurality of steroid hormone mixed calibrators, a plurality of steroid hormone mixed quality control products, a plurality of steroid hormone mixed isotope internal standards, a first extractant, a second extractant, a first resolvent, a second resolvent, and a buffer;

[0061] The first extracting agent is one or more of methyl tert-butyl ether and ethyl acetate; the second extracting agent is one or more of n-hexane, cyclohexane and n-heptane;

[0062] The first resolvent is 80% to 100% methanol aqueous solution; the second resolvent is 30% to 70% methanol aqueous solution;

[0063] Further, the buffer is 4-hydroxyethylpiperazineethanesulfonic acid, sodium phosphate, sodium borate buffer;

[0064] Further, the multiple steroid hormone calibrants are cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone sulfate; the multiple steroid hormone internal standards are cortisone-d8, cortisol-d4, corticosterone-d4, 11-deoxycortisol-d7, androstenedione-d7, testosterone-d3, dehydroepiandrosterone-d5, 17-hydroxyprogesterone-d8, progesterone-d9, 17-hydroxypregnenolone-d3, and dehydroepiandrosterone sulfate-d6;

[0065] Furthermore, the HPLC conditions are as follows: injector temperature: 4°C; column temperature: 50°C; run time: 5 min; gradient elution.

[0066]

[0067] The mobile phase A: 0-1 mM ammonium fluoride aqueous solution;

[0068] The mobile phase B: methanol

[0069] Furthermore, the mass spectrometry conditions are:

[0070] Positive ion mode: capillary voltage: 3.0 kV; negative ion mode: capillary voltage: -2.5 kV; desolvation temperature: 550°C; desolvation gas flow rate: 800 L / hr; cone gas flow rate: 150 L / hr; multiple reaction ion detection.

[0071] Furthermore, the content of the multiple steroid hormones is calculated as follows:

[0072] The calibration curve and regression equation were established with the concentration of the mixed steroid hormone calibrator as the horizontal axis and the ratio of the peak area measured between the mixed steroid hormone calibrator and the corresponding internal standard as the vertical axis. The peak area ratios of the measured multiple steroid hormones in serum specimens and the internal standard were substituted into the linear regression equation to calculate the concentrations of the multiple steroid hormones in serum specimens.

[0073] In Examples 1 to 16 and Verification Examples 1 to 6 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0074] The present invention will be further described below in conjunction with embodiments:

[0075] Example 1

[0076] A kit for simultaneously determining multiple steroid hormones in serum comprises the following components as shown in Table 1:

[0077] Table 1 Components of the kit for simultaneous determination of FT3 and FT4 in blood

[0078]

[0079]

[0080] In this embodiment, the calibrators of components 1 to 6 and the quality control materials of components 7 to 8 include: cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone sulfate; component 9 includes: cortisone-d8, cortisol-d4, corticosterone-d4, 11-deoxycortisol-d7, androstenedione-d7, testosterone-d3, dehydroepiandrosterone-d5, 17-hydroxyprogesterone-d8, progesterone-d9, 17-hydroxypregnenolone-d3, and dehydroepiandrosterone sulfate-d6.

[0081] Components 1 to 6 are mixed calibrators of various steroid hormones at different concentrations, which are used to draw calibration curves. The concentrations of cortisone calibrators are 200, 50, 10, 5, 1, and 0.5 ng / mL, respectively; the concentrations of cortisol calibrators are 500, 125, 25, 12.5, 2.5, and 1.25 ng / mL, respectively; the concentrations of corticosterone calibrators are 20, 5, 1, 0.5, 0.1, and 0.05 ng / mL, respectively; the concentrations of 11-deoxycortisol calibrators are 10, 2.5, 0.5, 0.25, 0.05, and 0.025 ng / mL, respectively; the concentrations of androstenedione calibrators are 10, 2.5, 0.5, 0.25, 0.05, and 0.025 ng / mL, respectively; the concentrations of testosterone calibrators are 10, 2.5, 0.5, 0.25, 0.05, and 0.025 ng / mL, respectively. The concentrations of the standard samples were 15, 3.75, 0.75, 0.375, 0.075, and 0.0375 ng / mL, respectively; the concentrations of the dehydroepiandrosterone calibrator were 200, 50, 10, 5, 1, and 0.5 ng / mL, respectively; the concentrations of the 17-hydroxyprogesterone calibrator were 20, 5, 1, 0.5, 0.1, and 0.05 ng / mL, respectively; the concentrations of the progesterone calibrator were 40, 10, 2, 1, 0.2, and 0.1 ng / mL, respectively; the concentrations of the 17-hydroxypregnenolone calibrator were 100, 25, 5, 2.5, 0.5, and 0.25 ng / mL, respectively; the concentrations of the dehydroepiandrosterone sulfate calibrator were 5000, 1250, 250, 125, 25, and 12.5 ng / mL, respectively.

[0082] Components 7 and 8 are mixed low-value and high-value quality control products of various steroid hormones, among which the concentrations of cortisone quality control products are 2 and 20 ng / mL respectively; the concentrations of cortisol quality control products are 5 and 50 ng / mL respectively; the concentrations of corticosterone quality control products are 0.2 and 2 ng / mL respectively; the concentrations of 11-deoxycortisol quality control products are 0.1 and 1 ng / mL respectively; the concentrations of androstenedione quality control products are 0.1 and 1 ng / mL respectively; the concentrations of testosterone quality control products are 0.15 and 1.5 ng / mL respectively; the concentrations of dehydroepiandrosterone quality control products are 2 and 20 ng / mL respectively; the concentrations of 17-hydroxyprogesterone quality control products are 0.2 and 2 ng / mL respectively; the concentrations of progesterone quality control products are 0.4 and 4 ng / mL respectively; the concentrations of 17-hydroxypregnenolone quality control products are 1 and 10 ng / mL respectively; and the concentrations of dehydroepiandrosterone sulfate quality control products are 50 and 500 ng / mL respectively.

[0083] Component 9 is a mixed stable internal standard of multiple steroid hormones, among which the concentration of cortisone-d8 is 15ng / mL; the concentration of cortisol-d4 is 100ng / mL; the concentration of corticosterone-d4 is 1ng / mL; the concentration of 11-deoxycortisol-d7 is 0.5ng / mL; the concentration of androstenedione-d7 is 1ng / mL; the concentration of testosterone-d3 is 0.75ng / mL; the concentration of dehydroepiandrosterone-d5 is 10ng / mL; the concentration of 17-hydroxyprogesterone-d8 is 2.5ng / mL; the concentration of progesterone-d9 is 2ng / mL; the concentration of 17-hydroxypregnenolone-d3 is 5ng / mL; and the concentration of dehydroepiandrosterone sulfate-d6 is 250ng / mL.

[0084] Example 2

[0085] A kit for simultaneously determining multiple steroid hormones in serum, wherein the first extractant in component 10 is ethyl acetate, and the other components are the same as those in Example 1.

[0086] Example 3

[0087] A kit for simultaneously determining multiple steroid hormones in serum, wherein the first extractant in component 10 is a 1:1 ratio of methyl tert-butyl ether to ethyl acetate, and the other components are the same as those in Example 1.

[0088] Verification Example 1

[0089] The kits obtained in Examples 1 to 3 can all extract multiple steroid hormones in serum. However, the signal intensities (extraction efficiencies) of multiple steroid hormones in serum with the same concentration are, from high to low, methyl tert-butyl ether > methyl tert-butyl ether: ethyl acetate is 1:1 > ethyl acetate.

[0090] Example 4

[0091] A kit for simultaneously determining multiple steroid hormones in serum, wherein the second extractant in component 11 is cyclohexane, and the other components are the same as those in Example 1.

[0092] Example 5

[0093] A kit for simultaneously determining multiple steroid hormones in serum, wherein the second extractant in component 11 is n-heptane, and the other components are the same as those in Example 1.

[0094] Verification Example 2

[0095] The kits obtained in Examples 1, 4 and 5 can effectively remove small polar impurities in the first reconstituted sample. At the same time, the signal-to-noise ratios (impurity removal capabilities) of multiple steroid hormones in serum with the same concentration are similar.

[0096] Example 6

[0097] A kit for simultaneously determining multiple steroid hormones in serum, wherein the other components are the same as those in Example 1 except that the first resolvent in component 12 is 80% methanol aqueous solution.

[0098] Example 7

[0099] A kit for simultaneously determining multiple steroid hormones in serum, wherein the components are the same as those in Example 1 except that the first resolvent in component 12 is 100% methanol.

[0100] Verification Example 3

[0101] The kits obtained in Examples 1, 6, and 7 can effectively dissolve multiple steroid hormones in the first extraction sample. However, the signal intensity (redissolution efficiency) of multiple steroid hormones in serum with the same concentration is from high to low, 90% methanol aqueous solution > 80% methanol aqueous solution > 100% methanol.

[0102] Example 8

[0103] A kit for simultaneously determining multiple steroid hormones in serum, wherein the other components are the same as those in Example 1 except that the second resolvent in component 13 is a 30% methanol aqueous solution.

[0104] Example 9

[0105] A kit for simultaneously determining multiple steroid hormones in serum, wherein the other components are the same as those in Example 1 except that the second resolvent in component 13 is a 50% methanol aqueous solution.

[0106] Example 10

[0107] A kit for simultaneously determining multiple steroid hormones in serum, wherein the other components are the same as those in Example 1 except that the second resolvent in component 13 is a 60% methanol aqueous solution.

[0108] Embodiment 11

[0109] A kit for simultaneously determining multiple steroid hormones in serum, wherein the other components are the same as those in Example 1 except that the second resolvent in component 13 is a 70% methanol aqueous solution.

[0110] Verification Example 4

[0111] The kits obtained in Example 1 and Examples 8 to 11 can effectively dissolve multiple steroid hormones in the second extraction sample. However, the signal intensity (redissolution efficiency) of multiple steroid hormones in serum with the same concentration is from high to low: 40% methanol aqueous solution ≈ 50% methanol aqueous solution > 60% methanol aqueous solution (slightly worse peak shape) > 70% methanol aqueous solution (not very good peak shape) > 30% methanol aqueous solution.

[0112] Example 12

[0113] A kit for simultaneously determining multiple steroid hormones in serum, wherein the buffer in component 14 is sodium phosphate buffer, and the other components are the same as those in Example 1.

[0114] Example 13

[0115] A kit for simultaneously determining multiple steroid hormones in serum, wherein the buffer in component 14 is a boric acid buffer, and the other components are the same as those in Example 1.

[0116] Verification Example 5

[0117] The kits obtained in Examples 1, 12, and 13 were used as blank matrices for the preparation of calibrators and quality control products, and similar test results were obtained.

[0118] Embodiment 14

[0119] A method for simultaneously determining multiple steroid hormones in serum, using the kit obtained in Example 1, comprises the following steps:

[0120] (1) Sample pretreatment:

[0121] 1) Extraction 1: Add 800 μL of the first extractant to 200 μL of serum specimen, 200 μL of mixed steroid hormone calibrator, and 200 μL of mixed steroid hormone quality control, respectively, and vortex and oscillate for 5 minutes;

[0122] 2) Centrifugation: centrifuge at 4°C and 12000g for 10 min, take 600 μL of supernatant to obtain the first extraction sample;

[0123] 3) Reconstitution 1: After drying with nitrogen, add 600 μL of the first reconstitution solvent and vortex for 5 min to obtain the first reconstituted sample;

[0124] 4) Extraction 2: Add 600 μL of the second extractant to the first reconstituted sample, and vortex and oscillate for 5 minutes;

[0125] 5) Centrifugation: Centrifuge at 4°C and 12000g for 10 min, discard the supernatant, and take 500 μL of the lower layer solution to obtain the second extraction sample;

[0126] 6) Reconstitution II: After drying with nitrogen, add 100 μL of the second reconstitution solvent and vortex for 5 min to obtain the second reconstituted sample;

[0127] 7) Detection: Centrifuge at 4°C and 12,000 g for 3 min, take 90 μL of the supernatant and place it in a 96-well injection plate or injection vial for liquid chromatography-tandem mass spectrometry analysis.

[0128] (2) High performance liquid chromatography tandem mass spectrometry detection:

[0129] 1) HPLC conditions:

[0130] Chromatographic column: AcquityUPLC BEH C18 (2.1×50 mm, 1.7 μm).

[0131] Column temperature: 50℃.

[0132] Flow rate: 0.3mL / min.

[0133] Mobile phase A (aqueous phase): 0.5 mM ammonium fluoride aqueous solution;

[0134] Mobile phase B (organic phase): methanol.

[0135] Gradient elution was used, and the gradient is shown in Table 2:

[0136] Table 2

[0137]

[0138]

[0139] 2) Mass spectrometry conditions:

[0140] Positive ion mode: capillary voltage: 3.0 kV; negative ion mode: capillary voltage: -2.5 kV; desolvation temperature: 550 °C; desolvation gas flow rate: 800 L / hr; cone gas flow rate: 150 L / hr;

[0141] Scanning mode: Multiple reaction monitoring. Specific parameters are shown in Table 3:

[0142] Table 3

[0143]

[0144] 3) Internal standard method calculation:

[0145] The internal standard method was used for quantification, with the concentration of various steroid hormones in a series of mixed calibrators as the horizontal axis (x), and the ratio of the peak area measured between the calibrators and the corresponding internal standards as the vertical axis (y), to establish a calibration curve and regression equation; the peak area ratios of various steroid hormones in serum specimens and internal standards were substituted into the linear regression equation to calculate the concentrations of various steroid hormones in serum specimens.

[0146] (3) The test results of the method of this embodiment are as follows:

[0147] 1) Quantitative limit: The lowest concentration of multiple steroid hormones (S6), with S / N ≥ 10, CV ≤ 20%, and relative deviation within ± 20%.

[0148] 2) Calibration curve: Within the linear range, the correlation coefficient of multiple steroid hormones is r≥0.99, the relative deviation of the lowest concentration calibration is within ±20%, and the relative deviation of the calibration at other concentration points is within ±15%.

[0149] 3) Precision test: The intra-day and inter-day precision CV of low-value and high-value mixed quality control products are ≤15%.

[0150] 4) Accuracy: The intra-day and inter-day accuracy (relative deviation) of low-value and high-value mixed quality control products are between ±15%.

[0151] 5) Sample recovery rate: The recovery rate of low-value and high-value mixed quality control products is between 85% and 115%.

[0152] Embodiment 15

[0153] A method for simultaneously determining multiple steroid hormones in serum is the same as Example 14 except that mobile phase A (aqueous phase) is pure water.

[0154] Example 16

[0155] A method for simultaneously determining multiple steroid hormones in serum is the same as Example 14 except that mobile phase A (aqueous phase): 1 mM ammonium fluoride aqueous solution.

[0156] Verification Example 6

[0157] Examples 14 to 16 can all detect multiple steroid hormones, but the signal intensity (redissolution efficiency) of multiple steroid hormones in serum with the same concentration is from high to low, 0.5 mM ammonium fluoride aqueous solution > 1 mM ammonium fluoride aqueous solution > 0 mM ammonium fluoride aqueous solution (pure water).

[0158] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a detection reagent or a detection kit comprising the detection reagent in the preparation of a product for detecting steroid hormones; The detection reagent comprises: Calibrators, quality controls, internal standards, extractants, resolvents and buffers; Among the extractants, the first extractant is methyl tert-butyl ether, and the second extractant is n-hexane; In the resolubilizing agent, the first resolubilizing agent is a 90% methanol aqueous solution, and the second resolubilizing agent is a 40% methanol aqueous solution; The calibrant comprises one or more of cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone or dehydroepiandrosterone sulfate; the buffer comprises one or more of 4-hydroxyethylpiperazineethanesulfonic acid, sodium phosphate or sodium borate buffer; The concentration of cortisone is 0.5-200ng / mL; the concentration of cortisol is 1.25-500ng / mL; the concentration of 11-deoxycortisol is 0.025-10ng / mL; the concentration of androstenedione is 0.025-10ng / mL; the concentration of testosterone is 0.0375-15ng / mL; the concentration of dehydroepiandrosterone is 0.5-200ng / mL; the concentration of 17-hydroxyprogesterone is 0.05-20ng / mL; the concentration of progesterone is 0.1-40ng / mL; the concentration of 17-hydroxypregnenolone is 0.25-100ng / mL; the concentration of hydrogen epiandrosterone sulfate is 12.5-5000ng / mL; The detection comprises taking the detection reagent or the detection kit and mixing it with the sample to be detected, and obtaining the concentration of the analyte in the sample to be detected after quantification; The quantification adopts high performance liquid chromatography tandem mass spectrometry; the conditions of high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry are: injector temperature is 4°C, column temperature is 50°C, running time is 5min, flow rate is 0.3mL / min, gradient elution; The procedure of the gradient elution is as follows: The mobile phase A is a 0-1 mM ammonium fluoride aqueous solution; the mobile phase B is methanol; The mass spectrometry conditions in the high performance liquid chromatography tandem mass spectrometry method are: the capillary voltage in the positive ion mode is 3.0 kV; the capillary voltage in the negative ion mode is -2.5 kV; the desolvation temperature is 550°C; the desolvation gas flow rate is 800 L / hr; the cone gas flow rate is 150 L / hr; and multiple reaction ion detection.

2. The use according to claim 1, characterized in that The mixing comprises the following steps: S1: Mix the sample to be tested with the calibrator, the quality control product, and the extractant, centrifuge, and collect the supernatant to obtain a first extraction sample; S2: taking the first extraction sample, concentrating it, mixing it with the resolvent, and then mixing it with the extractant, centrifuging it, and collecting the supernatant to obtain a second extraction sample; S3: After the second extraction sample is concentrated, it is mixed with the resolvent, centrifuged, and the supernatant is collected to obtain the sample to be tested.

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