CN115343398A - Kit and method for simultaneously measuring multiple steroid hormones in serum - Google Patents
Kit and method for simultaneously measuring multiple steroid hormones in serum Download PDFInfo
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- CN115343398A CN115343398A CN202211143975.0A CN202211143975A CN115343398A CN 115343398 A CN115343398 A CN 115343398A CN 202211143975 A CN202211143975 A CN 202211143975A CN 115343398 A CN115343398 A CN 115343398A
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Steroid Compounds (AREA)
Abstract
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. The present invention provides a detection reagent comprising: a calibrator, a quality control product, an internal standard product, an extractant, a double solvent and a buffer solution; the extraction agent comprises one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane; the double solvent comprises methanol water solution. The kit provided by the invention can achieve the purpose of simultaneously detecting the concentration of the multiple steroid hormones only by 200 mu L of serum samples, can accurately determine the content of the multiple steroid hormones with extremely low concentration (ng/mL level) in blood by only adopting a two-step extraction method, has the advantages of simple operation, sensitivity, accuracy and wide linear dynamic range (400 times) compared with the prior art for detecting the multiple steroid hormones, can simultaneously determine the multiple steroid hormones, and can be used for the conventional detection of the multiple steroid hormones in clinical serum.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a kit and a method for simultaneously measuring multiple steroid hormones in serum.
Background
Steroid hormones (Steroid hormons), also called Steroid hormones, are lipid-soluble small molecular hormones produced by the enzymatic metabolism of cholesterol through a series of enzymes, including mineralocorticoids, glucocorticoids and sex hormones, which are divided into androstane, estrane and pregnane according to chemical structures by taking cyclopentanoperhydrophenanthrene as a mother nucleus. Steroid hormones play an important role in maintaining life, regulating metabolism of body substances, promoting development of sexual organs, maintaining fertility and the like. Steroid hormones are mainly delivered through blood, act by binding to receptors in a very small dose, and have extremely high specificity. When the steroid hormone level in the body is disturbed, diseases such as congenital steroid metabolic disorder (e.g. congenital adrenal hyperplasia, congenital developmental disorder, congenital salt homeostasis disorder, etc.) and acquired steroid metabolic disorder (e.g. primary hyperaldosteronism, cushing's syndrome, addison's disease, hyperandrogenism and mental state disease) are caused.
Steroid hormones are low in content in organisms, similar in structure, various in types and large in concentration difference, are generally in the orders of nmol/L and pmol/L, and are different according to different ages and sexes, so that the accurate quantification of the steroid hormones has extremely high requirements on the sensitivity of instruments and the accuracy of a method.
At present, the detection method of steroid hormones mainly comprises a chemical immunoassay method, a gas chromatography-mass spectrometry combined technology and a liquid chromatography tandem mass spectrometry. Before the 20 th century, the chemical immunization method is widely applied in clinic due to simple and quick operation, but the traditional chemical immunization method has a plurality of defects: 1) The sensitivity is low, and when the concentration of a sample is very low, a large sample amount is required for enrichment and purification; 2) The applicability is poor, and the antibody of the object to be detected is easy to generate cross reaction with endogenous substances or metabolites with similar structures, so that a false positive result is caused; 3) The chemical immunity method has been gradually replaced by chromatography because the analysis efficiency is low, only 1 substance can be measured usually 1 time, and the qualitative and quantitative analysis of multiple hormones cannot be carried out simultaneously. The gas chromatography-mass spectrometry technology has high sensitivity, but often requires a complicated derivatization step and a long analysis time (generally more than or equal to 30 min), and currently lacks a derivatization reagent suitable for all steroid hormones. The liquid chromatography tandem mass spectrometry avoids the complicated derivatization step of gas chromatography, has strong specificity, can well eliminate the interference of substances with similar structures in the matrix, and is the most reliable detection method at present.
Therefore, the establishment of an analysis method capable of simultaneously and accurately measuring multiple steroid hormones has very important guiding significance for the diagnosis of clinical diseases.
Disclosure of Invention
In view of the above, the present invention provides a kit and a method for simultaneously measuring multiple steroid hormones in serum. The application provides a kit of multiple steroid hormone in simultaneous determination serum, including multiple steroid hormone mixed calibrator working solution, multiple steroid hormone mixed quality control article working solution, multiple steroid hormone mixed stable isotope internal standard solution, first extractant, second extractant, first double solvent, second double solvent, buffer solution, this scheme is easy and simple to handle, the specificity is high, the lot difference is little, sensitivity is high, dynamic range is wide, can satisfy clinical detection demand to multiple steroid hormone. The application also provides a method for simultaneously measuring multiple steroid hormones in serum, which does not need derivatization treatment, and simplifies the detection steps by extracting, extracting and purifying the sample through liquid-liquid extraction.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a detection reagent comprising: a calibrator, a quality control product, an internal standard product, an extractant, a double solvent and a buffer solution;
the extracting agent comprises one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane;
the double solvent comprises methanol water solution.
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.
In some embodiments of the invention, in the above-mentioned detection reagent, the quality control substance comprises one or more of cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone or dehydroepiandrosterone sulfate.
In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of cortisone is 2 and 20ng/mL; the concentration of the cortisol is 5 and 50ng/mL; the concentration of said corticosterone is 0.2 and 2ng/mL; the concentration of the 11-deoxycorticosterol is 0.1 and 1ng/mL; the concentration of the androstenedione is 0.1 and 1ng/mL; the testosterone concentration is 0.15 and 1.5ng/mL; the concentration of dehydroepiandrosterone is 2 and 20ng/mL; the concentration of the 17-hydroxyprogesterone is 0.2 and 2ng/mL; the concentration of the progesterone is 0.4 and 4ng/mL; the concentration of the 17-hydroxypregnanolone is 1 and 10ng/mL; the concentration of the dehydroepiandrosterone sulfate is 50 and 500ng/mL.
In some embodiments of the invention, in the above-described test reagents, the internal standard comprises one or more of cortisone-d 8, cortisol-d 4, corticosterone-d 4, 11-deoxycorticosterol-d 7, androstenedione-d 7, testosterone-d 3, dehydroepiandrosterone-d 5, 17-hydroxyprogesterone-d 8, progesterone-d 9, 17-hydroxyprogesterone-d 3, or dehydroepiandrosterone sulfate-d 6.
In some embodiments of the present invention, in the above detection reagent, the concentration of cortisone-d 8 is 15ng/mL; the concentration of the cortisol-d 4 is 100ng/mL; the concentration of the corticosterone-d 4 is 1ng/mL; the concentration of the 11-deoxycorticosterol-d 7 is 0.5ng/mL; the concentration of the androstenedione-d 7 is 1ng/mL; the concentration of testosterone-d 3 is 0.75ng/mL; the concentration of the dehydroepiandrosterone-d 5 is 10ng/mL; the concentration of the 17-hydroxyprogesterone-d 8 is 2.5ng/mL; the concentration of the progesterone-d 9 is 2ng/mL; the concentration of the 17-hydroxypregnanolone-d 3 is 5ng/mL; the concentration of the dehydroepiandrosterone-d 6 sulfate is 250ng/mL.
In some embodiments of the present invention, in the above detection reagent, the concentration of the methanol aqueous solution is 30% to 100%.
In some embodiments of the present invention, in the above-mentioned detection reagent, the concentration of the methanol aqueous solution is 30%, 50%, 60%, 70%, 80%, or 100%.
In some embodiments of the present invention, in the above-mentioned detection reagent, the calibrator comprises one or more of cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone, or dehydroepiandrosterone sulfate; the buffer solution comprises: 4-hydroxyethyl piperazine ethanesulfonic acid, sodium phosphate or sodium borate buffer solution.
In some embodiments of the present invention, in the above detection reagent, the concentration of cortisone is 0.5-200 ng/mL; the concentration of the cortisol is 1.25-500 ng/mL; the concentration of the 11-deoxycorticosterol is 0.025-10 ng/mL; the concentration of the androstenedione is 0.025-10 ng/mL; the concentration of the testosterone is 0.0375-15 ng/mL; the concentration of the dehydroepiandrosterone is 0.5-200 ng/mL; the concentration of the 17-hydroxyprogesterone is 0.05-20 ng/mL; the concentration of the progesterone is 0.1-40 ng/mL; the concentration of the 17-hydroxypregnanolone is 0.25-100 ng/mL; the concentration of the epiandrosterone sulfate is 12.5-5000 ng/mL.
In some embodiments of the present invention, in the above detection reagent, the concentration of cortisone is 200, 50, 10, 5, 1, 0.5ng/mL; the concentration of the cortisol is 500, 125, 25, 12.5, 2.5 and 1.25ng/mL; the concentration of the 11-deoxycorticosterol is 10, 2.5, 0.5, 0.25, 0.05 and 0.025ng/mL; the concentration of the androstenedione is 10, 2.5, 0.5, 0.25, 0.05 and 0.025ng/mL; the concentration of testosterone is 15, 3.75, 0.75, 0.375, 0.075 and 0.0375ng/mL; the concentration of dehydroepiandrosterone is 200, 50, 10, 5, 1, 0.5ng/mL, and the concentration of 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 and 0.1ng/mL; the concentration of the 17-hydroxypregnanolone is 100, 25, 5, 2.5, 0.5 and 0.25ng/mL; the concentration of the epiandrosterone sulfate is 5000, 1250, 250, 125, 25 and 12.5ng/mL.
The invention also provides a detection kit, which comprises the detection reagent and an acceptable auxiliary agent or carrier.
The invention also provides application of the detection reagent or the detection kit in preparation of products for detecting steroid hormones.
In some embodiments of the invention, in the above applications, the steroid hormone comprises: one or more of cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone or dehydroepiandrosterone sulfate.
In some embodiments of the present invention, in the above applications, the detecting includes mixing the detection reagent or the detection kit with a sample to be detected, and obtaining the concentration of the analyte in the sample to be detected after quantifying.
In some embodiments of the invention, in the above applications, the mixing comprises the steps of:
s1: mixing the sample to be detected with the calibrator, the quality control product and the extractant, centrifuging, and collecting a supernatant to obtain a first extraction sample;
s2: concentrating the first extraction sample, mixing with the double solvent, mixing with the extractant, centrifuging, and collecting a supernatant to obtain a second extraction sample;
s3: and (3) concentrating the second extraction sample, mixing with the double solvent, centrifuging, and collecting a supernatant to obtain the sample to be detected.
In some embodiments of the invention, in the above application, the quantifying employs high performance liquid chromatography tandem mass spectrometry; the conditions of the high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry are as follows: the temperature of the sample injector is 4 ℃, the column temperature is 50 ℃, the running time is 5min, the flow rate is 0.3mL/min, and gradient elution is carried out;
the procedure for the gradient elution was as follows:
(ii) a The mobile phase A is 0-1 mM ammonium chloride aqueous solution; the mobile phase B is methanol.
In some embodiments of the present invention, in the above application, the conditions of mass spectrometry in high performance liquid chromatography tandem mass spectrometry are: the capillary voltage in the positive ion mode is 3.0kV; the capillary tube voltage in the negative ion mode is-2.5 kV; the temperature of the desolventizing agent is 550 ℃; the gas flow rate for solvent removal is 800L/hr; the flow rate of the taper hole gas is 150L/hr; and (4) detecting multiple reactive ions.
In some embodiments of the invention, in the above application, the conditions of the mass spectrum are as follows:
in some embodiments of the invention, in the above applications, the quantification employs an internal standard method; and the internal standard method comprises the steps of taking the concentration of the steroid hormone in the calibrator as a horizontal coordinate (x), measuring the peak area ratio of the steroid hormone in the sample to be measured and the internal standard substance in the calibrator and the internal standard substance corresponding to the internal standard substance, substituting the peak area ratio into a linear regression equation, and calculating the concentration of the steroid hormone in the sample to be measured.
The present invention provides a detection reagent comprising: a calibrator, a quality control product, an internal standard product, an extractant, a double solvent and a buffer solution;
the extraction agent comprises one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane;
the double solvent comprises methanol water solution.
The beneficial effects of the invention include:
(1) The kit provided by the invention can achieve the purpose of simultaneously detecting the concentration of the multiple steroid hormones only by 200 mu L of serum samples, can accurately determine the content of the multiple steroid hormones with extremely low concentration (ng/mL level) in blood by only adopting a two-step extraction method, has the advantages of simple operation, sensitivity, accuracy and wide linear dynamic range (400 times) compared with the prior art for detecting the multiple steroid hormones, can simultaneously determine the multiple steroid hormones, and can be used for the conventional detection of the multiple steroid hormones in clinical serum.
(2) The kit provided by the invention is simple and convenient to operate, high in specificity, small in batch difference, high in sensitivity and wide in dynamic range, and can meet the clinical detection requirements on multiple steroid hormones.
(3) The method for simultaneously measuring the multiple steroid hormones in the serum provided by the application does not need derivatization treatment, and extracts and purifies the sample through liquid-liquid extraction, so that the detection steps are simplified.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below.
FIG. 1 shows LC-MS/MS chromatograms of a multi-steroid hormone calibrator;
FIG. 2 shows LC-MS/MS chromatograms of a multi-steroid hormone calibrator in a serum sample.
Detailed Description
The invention discloses a kit and a method for simultaneously measuring multiple steroid hormones in serum.
It should be understood that one or more of the expressions "\8230", individually include each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and/or" in connection with three or more of the stated objects shall be understood to have the same meaning unless otherwise understood from the context.
The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, are generally to be construed as open-ended and non-limiting, e.g., without excluding other unstated elements or steps, unless specifically stated otherwise or otherwise understood from context.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.
The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
In addition, the numerical ranges and parameters setting forth the invention are approximations, and the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless expressly stated otherwise, it is understood that all ranges, amounts, values and percentages used in this disclosure are by weight of the "about" modification. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.
The invention provides a method for simultaneously measuring multiple steroid hormones in serum, which uses a kit and comprises the following steps:
s1: respectively adding the mixed calibrator working solution and the mixed quality control working solution into a blank matrix to prepare a calibration curve sample and a quality control sample;
s2: extracting multiple steroid hormones in the serum sample, the calibration curve sample and the quality control sample by using a first extracting agent, centrifuging and taking a supernatant to obtain a first extracted sample;
s3: drying and concentrating the first extraction sample by using nitrogen, and dissolving the first extraction sample by using a first double solvent to obtain a first double-dissolved sample;
s4: extracting impurities in the first redissolved sample by using a second extracting agent, centrifuging, then abandoning the supernatant, and taking the lower layer solution to obtain a second extracted sample;
s5: drying and concentrating a second extraction sample by using nitrogen, dissolving the second extraction sample by using a second double solvent, and detecting by using high performance liquid chromatography-tandem mass spectrometry;
the invention also provides a kit, which comprises the following components: the kit comprises a multi-steroid hormone mixed calibrator, a multi-steroid hormone mixed quality control product, a multi-steroid hormone mixed isotope internal standard, a first extracting agent, a second extracting agent, a first double solvent, a second double solvent and a buffer solution;
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;
the first double solvent is 80-100% methanol water solution; the second double solvent is 30-70% methanol water solution;
further, the buffer solution is 4-hydroxyethyl piperazine ethanesulfonic acid, sodium phosphate and sodium borate buffer solution;
further, the multi-steroid hormone calibrator is cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate; the multi-steroid hormone internal standard substance is cortisone-d 8, cortisol-d 4, corticosterone-d 4, 11-deoxycorticosterol-d 7, androstenedione-d 7, testosterone-d 3, dehydroepiandrosterone-d 5, 17-hydroxyprogesterone-d 8, progesterone-d 9, 17-hydroxyprogesterone-d 3 and dehydroepiandrosterone sulfate-d 6;
further, the high performance liquid chromatography conditions are as follows: sample injector temperature: 4 ℃; column temperature: 50 ℃; operating time: 5min; gradient elution, conditions as follows:
the mobile phase A:0 to 1mM ammonium fluoride aqueous solution;
the mobile phase B: methanol
Further, the mass spectrum conditions are as follows:
positive ion mode: capillary voltage: 3.0kV; negative ion mode: capillary voltage: -2.5kV; desolventizing temperature: 550 ℃; desolventizing air flow rate: 800L/hr; taper hole air flow velocity: 150L/hr; and (4) detecting multiple reactive ions.
Further, the content calculation method of the multiple steroid hormones comprises the following steps:
taking the concentration of the multi-steroid hormone mixed calibrator as a horizontal coordinate, taking the ratio of peak areas measured by the multi-steroid hormone mixed calibrator and corresponding internal standard substances as a vertical coordinate, and establishing a calibration curve and a regression equation; substituting the peak area ratios of the multiple steroid hormones in the serum sample and the internal standard substance into a linear regression equation to calculate the concentration of the multiple steroid hormones in the serum sample.
In examples 1 to 16 and verification examples 1 to 6 of the present invention, the raw materials and reagents used were all commercially available.
The invention is further illustrated by the following examples:
example 1
A kit for simultaneously measuring multiple steroid hormones in serum comprises the following components as shown in Table 1:
TABLE 1 Components of kit for simultaneous determination of FT3 and FT4 in blood
In this embodiment, the calibrator and the quality control materials of components 1 to 6 and 7 to 8 include: cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate; the component 9 comprises: cortisone-d 8, cortisol-d 4, corticosterone-d 4, 11-deoxycorticosterol-d 7, androstenedione-d 7, testosterone-d 3, dehydroepiandrosterone-d 5, 17-hydroxyprogesterone-d 8, progesterone-d 9, 17-hydroxyprogesterone-d 3, and dehydroepiandrosterone-d 6 sulfate.
The components 1-6 are mixed calibrators of multiple steroid hormones with different concentrations and are used for drawing a calibration curve, wherein the concentrations of the cortisone calibrators are respectively 200ng/mL, 50ng/mL, 10ng/mL, 5ng/mL, 1ng/mL and 0.5ng/mL; the concentrations of the cortisol calibrator are respectively 500, 125, 25, 12.5, 2.5 and 1.25ng/mL; the concentrations of the corticosterone calibrator are respectively 20, 5, 1, 0.5, 0.1 and 0.05ng/mL; the concentrations of 11-deoxycorticosterol calibrator are respectively 10, 2.5, 0.5, 0.25, 0.05 and 0.025ng/mL; the concentrations of androstenedione calibrator are respectively 10, 2.5, 0.5, 0.25, 0.05 and 0.025ng/mL; the concentrations of the testosterone calibrator are respectively 15, 3.75, 0.75, 0.375, 0.075 and 0.0375ng/mL; the concentrations of dehydroepiandrosterone calibrator are 200, 50, 10, 5, 1 and 0.5ng/mL respectively; the concentrations of the 17-hydroxyprogesterone calibrator are respectively 20, 5, 1, 0.5, 0.1 and 0.05ng/mL; the concentrations of the progesterone calibrator are respectively 40, 10, 2, 1, 0.2 and 0.1ng/mL; the concentrations of the 17-hydroxypregnanolone calibrator are respectively 100, 25, 5, 2.5, 0.5 and 0.25ng/mL; the concentrations of the dehydroepiandrosterone sulfate calibrator were 5000, 1250, 250, 125, 25, and 12.5ng/mL, respectively.
The components 7 and 8 are mixed low-value and high-value quality control products of multiple steroid hormones, wherein the concentration of the cortisone quality control products is 2ng/mL and 20ng/mL respectively; the concentration of the cortisol quality control substance is 5ng/mL and 50ng/mL respectively; the concentrations of the quality control substances of the corticosterone are respectively 0.2 ng/mL and 2ng/mL; the concentrations of the 11-deoxycorticosterol quality control substances are 0.1ng/mL and 1ng/mL respectively; the concentration of the androstenedione quality control product is 0.1ng/mL and 1ng/mL respectively; the concentration of the testosterone quality control substance is 0.15 ng/mL and 1.5ng/mL respectively; the concentrations of the dehydroepiandrosterone quality control substances are respectively 2ng/mL and 20ng/mL; the concentrations of the 17-hydroxyprogesterone quality control substances are respectively 0.2 ng/mL and 2ng/mL; the concentration of the progesterone quality control substance is 0.4 and 4ng/mL respectively; the concentrations of the 17-hydroxypregnanolone quality control substances are respectively 1ng/mL and 10ng/mL; the concentration of the quality control product of dehydroepiandrosterone sulfate is 50ng/mL and 500ng/mL respectively.
The component 9 is a stable internal standard substance mixed with multiple steroid hormones, wherein the concentration of cortisone-d 8 is 15ng/mL; the concentration of the cortisol-d 4 is 100ng/mL; the concentration of corticosterone-d 4 is 1ng/mL; the concentration of 11-deoxycorticosterol-d 7 is 0.5ng/mL; the concentration of androstenedione-d 7 is 1ng/mL; the concentration of testosterone-d 3 is 0.75ng/mL; the concentration of dehydroepiandrosterone-d 5 is 10ng/mL; the concentration of the 17-hydroxyprogesterone-d 8 is 2.5ng/mL; the concentration of the progesterone-d 9 is 2ng/mL; the concentration of the 17-hydroxypregnanolone-d 3 is 5ng/mL; the concentration of dehydroepiandrosterone-d 6 sulfate was 250ng/mL.
Example 2
A kit for simultaneously measuring multiple steroid hormones in serum is the same as that in example 1 except that the first extracting agent in the component 10 is ethyl acetate.
Example 3
A kit for simultaneously measuring multiple steroid hormones in serum comprises a first extracting agent in a component 10, namely methyl tert-butyl ether: ethyl acetate was 1.
Verification example 1
The kits obtained in examples 1 to 3 can extract various steroid hormones from serum, but the signal intensity (extraction efficiency) of various steroid hormones from serum at the same concentration was from high to low, and methyl t-butyl ether > methyl t-butyl ether: ethyl acetate is 1.
Example 4
A kit for simultaneously measuring multiple steroid hormones in serum is the same as that in example 1 except that the second extracting agent in the component 11 is cyclohexane.
Example 5
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the second extraction agent in component 11 is n-heptane.
Verification example 2
The kits obtained in examples 1, 4 and 5 can effectively remove small polar impurities in the first reconstituted sample, and simultaneously, the signal-to-noise ratios (impurity removal capabilities) of multiple steroid hormones in serum with the same concentration are similar.
Example 6
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the first double solvent in component 12 is 80% methanol aqueous solution.
Example 7
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the first double solvent in component 12 is 100% methanol.
Verification example 3
The kits obtained in examples 1, 6 and 7 were all effective in solubilizing the steroid hormones in the first extraction sample, but the signal intensities (reconstitution efficiencies) of the steroid hormones in the same serum concentration were from high to low, in this order of 90% methanol aqueous solution >80% methanol aqueous solution >100% methanol.
Example 8
A kit for simultaneously measuring the multiple steroid hormones in serum comprises the same components as in example 1 except that the second double solvent in component 13 is 30% methanol aqueous solution.
Example 9
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the second double solvent in component 13 is 50% methanol aqueous solution.
Example 10
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the second double solvent in component 13 is 60% methanol aqueous solution.
Example 11
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the second double solvent in component 13 is 70% methanol aqueous solution.
Verification example 4
The kits obtained in example 1 and examples 8 to 11 were each capable of efficiently dissolving a plurality of steroid hormones in the second extract sample, but the signal intensity (reconstitution efficiency) of a plurality of steroid hormones in the same concentration of serum was from high to low, and 40% methanol aqueous solution ≈ 50% methanol aqueous solution >60% methanol aqueous solution (slightly inferior peak profile) >70% methanol aqueous solution (less peak profile) >30% methanol aqueous solution.
Example 12
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the buffer solution in the component 14 is a sodium phosphate buffer solution.
Example 13
A kit for simultaneously measuring multiple steroid hormones in serum comprises the same components as in example 1 except that the buffer solution in component 14 is a boric acid buffer solution.
Verification example 5
The kits obtained in examples 1, 12 and 13 are used as blank substrates for preparing calibrator and quality control materials, and similar detection results are obtained.
Example 14
A method for simultaneously measuring multiple steroid hormones in serum, using the kit obtained in example 1, comprising the steps of:
(1) Sample pretreatment:
1) Extraction I: adding 800 μ L of first extractant into 200 μ L of serum sample, 200 μ L of multi-steroid hormone mixed calibrator, and 200 μ L of multi-steroid hormone mixed quality control respectively, and performing vortex oscillation extraction for 5min;
2) Centrifuging: centrifuging at 4 deg.C and 12000g for 10min, and collecting 600 μ L supernatant to obtain first extract sample;
3) Redissolving one: drying by using nitrogen, adding 600 mu L of first compound solvent, and performing vortex oscillation for 5min to obtain a first compound sample;
4) And (2) extraction II: respectively adding 600 mu L of second extracting agent into the first redissolved sample, and performing vortex oscillation extraction for 5min;
5) Centrifuging: centrifuging at 4 deg.C and 12000g for 10min, removing supernatant, and collecting 500 μ L lower layer solution to obtain second extraction sample;
6) And (2) redissolving: drying by using nitrogen, adding 100 mu L of second compound solvent, and performing vortex oscillation for 5min to obtain a second compound sample;
7) And (3) detection: centrifuging at 12000g for 3min at 4 deg.C, collecting 90 μ L supernatant, and performing liquid chromatography tandem mass spectrometry in 96-well sample inlet plate or sample inlet vial.
(2) High performance liquid chromatography tandem mass spectrometry detection:
1) High performance liquid chromatography conditions:
a chromatographic column: acquity UPLC BEH C18 (2.1X 50mm,1.7 μm).
Column temperature: at 50 deg.C.
Flow rate: 0.3mL/min.
Mobile phase a (aqueous phase): 0.5mM ammonium fluoride in water;
mobile phase B (organic phase): methanol.
Gradient elution was used, the gradient being shown in table 2:
TABLE 2
2) Mass spectrum conditions:
positive ion mode: capillary voltage: 3.0kV; negative ion mode: capillary voltage: -2.5kV; desolventizing temperature: 550 ℃; desolventizing air flow rate: 800L/hr; taper hole air flow rate: 150L/hr;
scanning mode: and (5) monitoring multiple reactions. The specific parameters are shown in table 3:
TABLE 3
3) Calculating by an internal standard method:
quantifying by adopting an internal standard method, taking the concentration of multiple steroid hormones in the series of mixed calibrators as a horizontal coordinate (x), taking the ratio of the peak area measured by the calibrators and the corresponding internal standard substance as a vertical coordinate (y), and establishing a calibration curve and a regression equation; substituting the peak area ratios of the multiple steroid hormones in the serum sample and the internal standard substance into a linear regression equation to calculate the concentrations of the multiple steroid hormones in the serum sample.
(3) The method of the embodiment has the following detection results:
1) Lower limit of quantitation: the minimum concentration (S6) of the steroid hormones is more than or equal to 10 percent, CV is less than or equal to 20 percent, and the relative deviation is within +/-20 percent.
2) Calibration curve: within the linear range, the correlation coefficient r of the multiple steroid hormones is more than or equal to 0.99, the relative deviation of the calibrator at the lowest concentration is within +/-20%, and the relative deviation of the calibrators at the other concentration points is within +/-15%.
3) And (3) precision test: the precision CV of the low-value and high-value mixed quality control product in the day and the daytime is less than or equal to 15 percent.
4) Accuracy: the day-to-day and day-to-day accuracy (relative deviation) of the low-value and high-value mixed quality controls was between ± 15%.
5) And (3) sample recovery rate: the recovery rate of the low-value and high-value mixed quality control products is between 85 and 115 percent.
Example 15
A method for simultaneously measuring multiple steroid hormones in serum comprises the following steps except a mobile phase A (water phase): the procedure of example 14 was repeated except for using pure water.
Example 16
A method for simultaneously measuring multiple steroid hormones in serum comprises the following steps except a mobile phase A (water phase): 1mM ammonium fluoride aqueous solution, the rest being the same as in example 14.
Verification example 6
Examples 14 to 16 all detected a large number of steroid hormones, but the signal intensity (reconstitution efficiency) of the steroid hormones in the same concentration serum was from high to low, 0.5mM ammonium fluoride aqueous solution >1mM ammonium fluoride aqueous solution >0mM ammonium fluoride aqueous solution (pure water).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A detection reagent, comprising: a calibrator, a quality control product, an internal standard product, an extractant, a double solvent and a buffer solution;
the extraction agent comprises one or more of methyl tert-butyl ether, ethyl acetate, n-hexane, cyclohexane or n-heptane;
the double solvent comprises methanol water solution.
2. The detection reagent of claim 1, wherein the concentration of the aqueous methanol solution is 30% to 100%.
3. The test reagent of claim 1 or 2, wherein the calibrator comprises one or more of cortisone, cortisol, corticosterone, 11-deoxycorticosterol, androstenedione, testosterone, dehydroepiandrosterone, 17-hydroxyprogesterone, progesterone, 17-hydroxyprogesterone, or dehydroepiandrosterone sulfate; the buffer solution comprises: one or more of 4-hydroxyethyl piperazine ethanesulfonic acid, sodium phosphate or sodium borate buffer.
4. The detection reagent of claim 3, wherein the concentration of cortisone is 0.5-200 ng/mL; the concentration of the cortisol is 1.25-500 ng/mL; the concentration of the 11-deoxycorticosterol is 0.025-10 ng/mL; the concentration of the androstenedione is 0.025-10 ng/mL; the concentration of the testosterone is 0.0375-15 ng/mL; the concentration of the dehydroepiandrosterone is 0.5-200 ng/mL; the concentration of the 17-hydroxyprogesterone is 0.05-20 ng/mL; the concentration of the progesterone is 0.1-40 ng/mL; the concentration of the 17-hydroxypregnanolone is 0.25-100 ng/mL; the concentration of the hydrogen epiandrosterone sulfate is 12.5-5000 ng/mL.
5. A test kit comprising a test reagent according to any one of claims 1 to 4 and an acceptable adjuvant or carrier.
6. Use of a detection reagent according to any one of claims 1 to 4 or a detection kit according to claim 5 in the manufacture of a product for the detection of a steroid hormone.
7. The use of claim 6, wherein said detecting comprises mixing said detection reagent or said detection kit with a sample to be tested, and obtaining the concentration of the analyte in said sample to be tested after quantification.
8. The use of claim 7, wherein the mixing comprises the steps of:
s1: mixing the sample to be detected with the calibrator, the quality control product and the extractant, centrifuging, and collecting a supernatant to obtain a first extraction sample;
s2: concentrating the first extraction sample, mixing the concentrated first extraction sample with the double solvent, mixing the concentrated first extraction sample with the extractant, centrifuging the mixture, and collecting supernatant to obtain a second extraction sample;
s3: and (3) concentrating the second extraction sample, mixing with the double solvent, centrifuging, and collecting the supernatant to obtain the sample to be detected.
9. The use of claim 7 or 8, wherein said quantification is by high performance liquid chromatography tandem mass spectrometry; the conditions of the high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry are as follows: the temperature of the sample injector is 4 ℃, the column temperature is 50 ℃, the running time is 5min, the flow rate is 0.3mL/min, and gradient elution is carried out;
the procedure for the gradient elution was as follows:
the mobile phase A is 0-1 mM ammonium chloride aqueous solution; the mobile phase B is methanol.
10. The use of claim 9, wherein the conditions for mass spectrometry in hplc tandem mass spectrometry are: the capillary voltage in the positive ion mode is 3.0kV; the capillary voltage in the negative ion mode is-2.5 kV; the temperature of the desolventizing agent is 550 ℃; the gas flow rate for solvent removal is 800L/hr; the flow rate of the taper hole gas is 150L/hr; and (4) detecting multiple reactive ions.
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