CN118604217A - Kit and method for enriching, concentrating and/or detecting PCOS-related steroid hormone - Google Patents
Kit and method for enriching, concentrating and/or detecting PCOS-related steroid hormone Download PDFInfo
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- CN118604217A CN118604217A CN202411074804.6A CN202411074804A CN118604217A CN 118604217 A CN118604217 A CN 118604217A CN 202411074804 A CN202411074804 A CN 202411074804A CN 118604217 A CN118604217 A CN 118604217A
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- 238000012795 verification Methods 0.000 description 1
- 230000001792 virilizing effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Abstract
The invention provides a kit and a method for enriching, concentrating and/or detecting PCOS related steroid hormone, and relates to the technical field of analysis and detection. After mixing humic acid with magnetic particles SiO 2@Fe3O4, carrying out pyrolysis treatment for 0.5-1.5 h at the temperature of 300-900 ℃ to obtain magnetic particles with the surface of an aromatic carbonaceous phase containing oxygen-containing functional groups, wherein the surface of the magnetic solid phase material is hydrophilic and lipophilic and amphiphilic, and has good enrichment and separation effects on small molecular organic matters; the kit also comprises an activating solution, a balancing solution, a dissociation solution, a first eluent, a second eluent and an eluent, and the kit can be used for preprocessing a sample to improve the accuracy of steroid hormone detection.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a kit and a method for enriching, concentrating and/or detecting PCOS related steroid hormone.
Background
The following statements merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting women of about 1/15 of their child bearing age. Its symptoms are diverse and mainly include hyperandrogenism (hirsutism and acne), irregular menstruation and metabolic dysfunction (obesity, insulin resistance, acanthosis nigricans). Polycystic ovary syndrome is also one of the most common causes of female infertility. It is estimated that 75% of PCOS patients remain undiagnosed, mainly because of the diversity of symptoms and the non-uniformity of clinical diagnostic criteria. More than one third of PCOS women spent at least 2 years before diagnosing the disease and at least three medical professionals were consulted. Diagnosis of PCOS is not a single standard or test method, and is based primarily on the exclusion of diseases with similar symptoms. The most common method of diagnosing PCOS today is to define PCOS according to at least 2 of 3 criteria: (1) hyperandrogens; (2) ovulation dysfunction; (3) polycystic ovary morphology (PCOM). Endocrine disorders of many steroid hormones (e.g., diagnosing cushing's syndrome, non-classical CAH, virilizing tumors, hyperandrogenism, etc.) also meet the criteria for diagnosing PCOS. Thus, PCOS can only be diagnosed after other diseases are excluded.
Steroid hormones mainly exist in a protein binding mode, and human blood contains a great amount of high-affinity binding proteins, so that the composition is complex, the content difference of different hormones is large, more structural analogues and isomers exist, and mutual interference is easy. In order to realize accurate quantification, high requirements on sensitivity and accuracy are met, a proper sample pretreatment mode needs to be selected to enrich a target object, so that the sensitivity is improved, and meanwhile, matrix interference is eliminated or reduced. At present, a great deal of literature proves that the measurement result is inaccurate, the quantitative limit is high, the linear range is narrow and the like due to the interference of structural analogues and substrates when the immune method is used for measuring the steroid hormone; meanwhile, each immunization method can only measure one steroid hormone, and the detection time is long. Many obstacles are presented to the diagnostic method for PCOS removal, so that a simple, practical, economical, applicable, high-throughput and accurate quantitative detection method is urgently needed.
Currently, tandem mass spectrometry is used to measure multi-steroid hormones, and the following sample pretreatment methods are generally used for blood sample treatment: firstly, a sample is subjected to liquid-liquid extraction after protein precipitation, the solvent for liquid-liquid extraction is generally volatile organic reagents such as ethyl acetate, n-hexane, methyl tertiary butyl ether and the like, liquid leakage easily occurs in the process of removing supernatant fluid, so that the loss or pollution of the sample is caused, and the requirement on personnel is high; secondly, the sample is subjected to derivatization after protein precipitation and then is subjected to liquid-liquid extraction, so that the sensitivity of an object to be detected can be improved, but the operation is complicated, and after the derivatization of a plurality of analytes, double peaks which cannot be separated from a baseline exist, so that the quantitative integration result is inaccurate; thirdly, a solid-phase extraction method is used after the sample is subjected to protein precipitation, so that the operation steps are more, consumables such as a solid-phase extraction column are more expensive, and the detection cost is increased.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an amphiphilic magnetic solid phase material and application of the amphiphilic magnetic solid phase material in enrichment, concentration and/or detection of PCOS related steroid hormone so as to alleviate defects in steroid hormone detection in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, a kit for enriching, concentrating and/or detecting PCOS-related steroid hormones is provided, the kit comprising an amphiphilic magnetic solid phase material, an activating solution, a balancing solution, a dissociation solution, a first eluent, a second eluent and an eluent;
After mixing humic acid with magnetic particles SiO 2@Fe3O4, carrying out pyrolysis treatment at 300-900 ℃ for 0.5-1.5 h to obtain magnetic particles with surfaces being carbonaceous phases; the magnetic particles SiO 2@Fe3O4 are nano particles with core-shell structures, wherein SiO 2 coats Fe 3O4;
The activating solution comprises methanol;
The balance liquid comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-10%, and the ratio of the methanol is not 0;
The dissociation solution comprises an ammonium acetate-acetic acid buffer solution, and the pH range is 5-6;
the first leaching solution comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-30%, and the ratio of the methanol is not 0;
The second eluent comprises one or more of normal hexane, isohexane and cyclohexane;
the eluent comprises one or more of methanol, acetonitrile and isopropanol;
The PCOS-associated steroid hormone includes one or more of progesterone, 17 alpha-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone sulfate, 4-androstenedione, testosterone, 17 alpha-hydroxy pregnenolone, and dihydrotestosterone.
In a second aspect, there is also provided a method of sample pretreatment comprising enriching and/or concentrating PCOS-related steroid hormone in the sample using the amphiphilic magnetic solid phase material of the first aspect, or the kit of the first aspect;
Mixing the amphiphilic magnetic solid phase material subjected to activation treatment and balance treatment in sequence with a sample system, enabling the amphiphilic magnetic solid phase material to adsorb PCOS related steroid hormone in a sample, separating the amphiphilic magnetic solid phase material, mixing with a first leaching solution, separating the amphiphilic magnetic solid phase material, mixing with a second leaching solution, eluting with an eluent, and collecting the eluent, wherein the eluent contains the PCOS related steroid hormone in the sample for subsequent detection;
The activation treatment comprises mixing an activation solution with the amphiphilic magnetic solid phase material;
the balancing treatment comprises mixing the amphiphilic magnetic solid phase material subjected to the activation treatment with a balancing liquid;
the sample system comprises a sample, methanol and dissociation liquid;
the activating solution, the balancing solution, the dissociating solution, the first eluent, the second eluent and the eluent are all the activating solution, the balancing solution, the dissociating solution, the first eluent, the second eluent and the eluent in the first aspect.
In a third aspect, there is also provided a method of detecting PCOS-related steroid hormone, comprising enriching and/or concentrating PCOS-related steroid hormone in a sample using the amphiphilic magnetic solid phase material of the first aspect, and then analyzing the PCOS-related steroid hormone in the sample using liquid chromatography tandem mass spectrometry;
The liquid chromatography conditions included:
the chromatographic column adopts a C18 chromatographic column;
the mobile phase system comprises a mobile phase A and a mobile phase B;
The mobile phase A is an aqueous ammonium formate solution or an aqueous ammonium acetate solution, and contains no more than 0.001% v/v formic acid; the mobile phase B is methanol solution of ammonium formate or methanol solution of ammonium acetate, and the mobile phase B contains not more than 0.001% v/v formic acid.
Compared with the prior art, the invention has the following beneficial effects:
The invention enriches, concentrates and/or detects steroid hormones through amphiphilic magnetic solid phase extraction materials. The amphiphilic magnetic solid phase extraction material is prepared by using a one-pot method and takes SiO 2@Fe3O4 magnetic particles as a substrate, and the whole preparation process is simple, good in repeatability and environment-friendly (no organic solvent or harmful chemical substances are involved). The preparation method uses a large amount of available starting raw materials (silicon dioxide and humic acid) with low cost, and the obtained magnetic particles have moderate size and basically range from 1 μm to 50 μm. The humic acid is a substance containing macromolecular base structure and composed of a hydrophobic framework and rich hydrophilic groups, and can be converted into an aromatic carbonaceous phase containing oxygen-containing functional groups through simple pyrolysis treatment, so that a novel mixed mode carbonaceous Xiang Jituan is generated, and the hydrophilic and lipophilic substances are good. The obtained magnetic particles have good enrichment and separation effects on endogenous substance micromolecular organic matters, greatly reduce matrix effect in the steroid detection process, ensure the accuracy of a quantitative method and are beneficial to simultaneously detecting various PCOS related steroid hormones.
The invention optimizes the magnetic particle extraction process and the detection method of liquid chromatography tandem mass spectrometry, particularly optimizes the mobile phase and elution program, adopts the extremely low-concentration acidic solution which is used irregularly, obtains good separation effect, and does not use the ion pair reagent ammonium fluoride which is commonly used in steroid hormone liquid chromatography analysis. Avoiding a series of problems caused by using ammonium fluoride as the mobile phase. For example, when ammonium fluoride is used, the chromatographic column needs to be strictly mixed with a non-ammonium fluoride mobile phase, and an analysis system using the ammonium fluoride needs to use the non-acid mobile phase to flush a pipeline so as to avoid corrosion of the pipeline, and meanwhile, the service life of the chromatographic column can be reduced after long-term use. The restriction of the mobile phase greatly reduces the use efficiency of the whole liquid system, thereby increasing the use cost. The detection method of the PCOS related steroid hormone provided by the invention can realize high-specificity separation of various PCOS related steroid hormones in a short time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
Fig. 1 is a graph of progesterone residuals (top) and linear graphs (bottom) for the test method of example 2;
FIG. 2 is a progesterone chromatogram of the assay of example 2;
FIG. 3 is a graph of 17 a-hydroxyprogesterone residual error plot (upper) and linear plot (lower) for the assay of example 2;
FIG. 4 is a 17 a-hydroxyprogesterone chromatogram of the assay of example 2;
FIG. 5 is a graph of the residual 11-deoxycorticosterone profile (top) and linear profile (bottom) for the test method of example 2;
FIG. 6 is a chromatogram of 11-deoxycorticosterone from the assay of example 2;
FIG. 7 is an aldosterone residual plot (upper) and linear plot (lower) of the assay of example 2;
FIG. 8 is a chromatogram of aldosterone from the assay of example 2;
FIG. 9 is a graph of the residual 11-deoxycortisol profile (top) and linear plot (bottom) for the test method of example 2;
FIG. 10 is a chromatogram of 11-deoxycortisol for the assay of example 2;
FIG. 11 is a graph of cortisol residual plot (top) and linear plot (bottom) for the test method of example 2;
FIG. 12 is a cortisol chromatogram of the assay of example 2;
FIG. 13 is a graph of dehydroepiandrosterone residual plot (top) and linear plot (bottom) for the test method of example 2;
FIG. 14 is a dehydrogenated epiandrosterone chromatogram of the assay of example 2;
FIG. 15 is a graph of dehydroepiandrosterone-sulfate residual plot (top) and linear plot (bottom) for the test method of example 2;
FIG. 16 is a chromatogram of dehydroepiandrosterone-sulfate for the assay of example 2;
FIG. 17 is a graph of androstenedione residual error plot (top) and linear plot (bottom) for the test method of example 2;
FIG. 18 is a chromatogram of androstenedione from the assay of example 2;
fig. 19 is a graph of testosterone residuals (top) and linear plots (bottom) for the test method of example 2;
FIG. 20 is a testosterone chromatogram of the assay of example 2;
FIG. 21 is a graph of pregnenolone residual error plot (top) and linear plot (bottom) for the detection method of example 2;
FIG. 22 is a pregnenolone chromatogram of the detection method of example 2;
FIG. 23 is a graph of the residual 17-hydroxy pregnenolone graph (top) and the linear graph (bottom) for the assay of example 2;
FIG. 24 is a 17-hydroxy pregnenolone chromatogram of the assay of example 2;
Fig. 25 is a graph of the dihydrotestosterone residual profile (top) and the linear profile (bottom) for the test method of example 2;
FIG. 26 is a chromatograms of dihydrotestosterone of the assay of example 2;
Fig. 27 is a chromatograms of dihydrotestosterone using mobile phase number 1 in example 5;
FIG. 28 IS a 17-hydroxy pregnenolone-IS chromatogram of example 5 with mobile phase number 1;
fig. 29 is a chromatograms of dihydrotestosterone using mobile phase number 2 in example 5;
FIG. 30 IS a 17-hydroxy pregnenolone-IS chromatogram of example 5 with mobile phase number 2;
fig. 31 is a chromatograms of dihydrotestosterone using mobile phase number 3 in example 5;
FIG. 32 IS a 17-hydroxy pregnenolone-IS chromatogram of example 5 with mobile phase number 3.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In a first aspect, a kit for enriching, concentrating and/or detecting PCOS-related steroid hormones is provided.
The PCOS-associated steroid hormone includes one or more of progesterone, 17 alpha-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone sulfate, 4-androstenedione, testosterone, 17 alpha-hydroxy pregnenolone, and dihydrotestosterone.
In alternative embodiments, the PCOS-related steroid hormone comprises progesterone, 17 a-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone sulfate, 4-androstenedione, testosterone, 17 a-hydroxy pregnenolone, and dihydrotestosterone.
The kit for enriching, concentrating and/or detecting the PCOS-related steroid hormone comprises the following components:
An amphiphilic magnetic solid phase material.
After mixing humic acid with magnetic particles SiO 2@Fe3O4, carrying out pyrolysis treatment at 300-900 ℃ for 0.5-1.5 h to obtain magnetic particles with surfaces being carbonaceous phases; the magnetic particles SiO 2@Fe3O4 are nano particles with core-shell structures, wherein SiO 2 coats Fe 3O4. Humic acid on the surface of the magnetic particle is pyrolyzed to form a carbonaceous mesophase composed of a hydrophobic framework and rich hydrophilic groups. The temperature of the pyrolysis treatment may be, for example, but not limited to, 300, 400, 500, 600, 700, 800, or 900 ℃, and the time of the pyrolysis treatment may be, for example, but not limited to, 0.5, 1, or 1.5 hours.
In an alternative embodiment, the temperature of the pyrolysis treatment is 600 ℃ and the time of the pyrolysis treatment is 1 h.
In an alternative embodiment, the mass ratio of the humic acid to the SiO 2@Fe3O4 magnetic particles is 0.05-0.5, for example, but not limited to, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, preferably 0.1.
In an alternative embodiment, the particle size of the amphiphilic magnetic solid phase material is 1-50 μm, for example, but not limited to 1, 5, 10, 20, 30 or 50 μm.
In an alternative embodiment, the preparation method comprises dissolving humic acid in water in a reaction vessel, then adding a formula amount of SiO 2@Fe3O4 magnetic particles, stirring the suspension, and then removing water; and (3) carrying out pyrolysis treatment on the obtained solid in nitrogen flow for 0.5-1.5 h at the temperature of 300-900 ℃, converting SiO 2@Fe3O4 modified by humic acid into hydrophilic-lipophilic balanced carbonaceous phase magnetic particles, and washing the magnetic particles with water until the washing waste liquid is neutral.
In an alternative embodiment, the mass ratio of the humic acid to the SiO 2@Fe3O4 magnetic particles is 0.05-0.5, for example, but not limited to, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, preferably 0.1.
In an alternative embodiment, the concentration of the humic acid aqueous solution is 1-5 g/L, preferably 2 g/L.
In an alternative embodiment, a rotary evaporator is used to remove moisture.
In an alternative embodiment, the pyrolysis treatment is performed in an alumina combustion boat within a quartz tube.
The SiO 2@Fe3O4 magnetic particles can be prepared by any method known in the art, and the invention is not limited thereto.
In an alternative embodiment, the SiO 2@Fe3O4 magnetic particles are prepared using a sol gel process.
In an alternative embodiment, the sol-gel process comprises suspending Fe 3O4 powder in purified water and sonicating, followed by adding cetyltrimethylammonium bromide (CTAB) powder and Triethanolamine (TEOA), and continuously stirring the mixture at a temperature of 70-90 ℃ for 0.5-1.5 h. And filtering and recycling to obtain SiO 2@Fe3O4 particles, washing and drying, and calcining at 500-580 ℃ for 6-8 hours to obtain SiO 2@Fe3O4 magnetic particles.
In an alternative embodiment, the ultrasonic time is 20-40 min, preferably 30 min.
In an alternative embodiment, the molar ratio of Fe 3O4, cetyltrimethylammonium bromide and triethanolamine is (0.8-1.2): (1.5-2), preferably 1:1:1.5.
In alternative embodiments, fe 3O4, cetyltrimethylammonium bromide and triethanolamine are continuously stirred under conditions such as, but not limited to, 70, 75, 80, 85 or 90 ℃, preferably at 80 ℃.
In alternative embodiments, the duration of agitation may be, for example, but not limited to, 0.5, 1, or 1.5 h, preferably 1.5 h.
In an alternative embodiment, stirring is continued in a thermostatic water bath.
In an alternative embodiment, the SiO 2@Fe3O4 particles are recovered by washing with ethanol and filtration.
In an alternative embodiment, the drying includes drying at 55-65 ℃ for 20-30 hours, and the drying temperature may be, for example, but not limited to 55, 60 or 65 ℃; the drying time may be, for example, but not limited to, 20, 22, 24, 26, 28, or 30 h.
In an alternative embodiment, the drying comprises drying at 60 ℃ for 24 hours.
In alternative embodiments, the temperature of the calcination may be, for example, but not limited to, 500, 510, 520, 530, 540, 550, 560, 570, or 580 ℃, preferably 540 ℃.
In alternative embodiments, the calcination time may be, for example, but not limited to, 6, 6.5, 7, 7.5, or 8h, preferably 7 h.
(II) activating solution, balancing solution, dissociating solution, first eluent, second eluent and eluent:
The activating solution comprises methanol;
The balance liquid comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-10%, and the ratio of the methanol is not 0; the volume ratio of methanol to water may be, for example, but not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably the volume ratio of methanol to water is 5%.
The dissociation solution comprises an ammonium acetate-acetic acid buffer solution, and the pH range is 5-6, preferably 5.5.
The first leaching solution comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-30%, and the ratio of the methanol is not 0; the volume ratio of methanol to water may be, for example, but not limited to, 1%, 5%, 10%, 15%, 20%, 25% or 30%, preferably the volume ratio of methanol to water is 15%.
The second eluent comprises one or more of n-hexane, isohexane and cyclohexane, preferably n-hexane.
The eluent comprises one or more of methanol, acetonitrile and isopropanol.
In an alternative embodiment, the kit further comprises: (iii) one or more of a calibrator, a quality control, an internal standard, and a blank matrix.
In an alternative embodiment, the blank substrate is a biological substrate comprising an aqueous solution of serum or serum albumin, the biological substrate being pretreated to remove endogenous steroid hormones. The removal refers to the fact that the content of endogenous steroid hormones in serum is lower than a detection threshold.
In an alternative embodiment, the pretreatment to remove endogenous steroid hormones comprises adsorbing endogenous steroid hormones in a biological matrix with a macroporous adsorbent resin.
In an alternative embodiment, the calibrator is formulated from the blank matrix.
In an alternative embodiment, the quality control is formulated from the blank matrix.
The calibration material and/or the quality control material prepared by using the blank matrix can simulate the real matrix environment, reduce the influence of matrix effect and improve the quantitative accuracy.
In an alternative embodiment, the macroporous adsorbent resin is a nonpolar macroporous adsorbent resin or a medium polarity macroporous adsorbent resin. The nonpolar macroporous adsorption resin is resin which is prepared by polymerizing monomers with small dipole moment and has no functional group and strong hydrophobicity on the pore surface. The medium polarity macroporous adsorption resin refers to an adsorption resin containing ester groups, and the surface hydrophobic part and the hydrophilic part coexist.
Aiming at the problems of complicated operation and long time consumption of preparing blank serum by activated carbon adsorption, activated carbon is easy to remain in a matrix solution after adsorption and is extremely difficult to completely separate, a high polymer adsorbent with large particles, stable structure and low price is preferably adopted, the pH of a biological matrix is not required to be regulated, endogenous micromolecules of the biological matrix can be directly adsorbed, the whole operation process is simple and rapid, the adsorbent can be completely separated from the biological matrix after adsorption, the biological matrix cannot be greatly influenced, and a prepared calibrator or quality control product can better simulate the biological matrix, so that the accuracy of a quantitative result is improved.
In an alternative embodiment, the pretreatment to remove endogenous steroid hormones comprises:
(a) Activating: placing a proper amount of macroporous adsorption resin in a container, adding ethanol for soaking, and separating the macroporous adsorption resin; and washing with ethanol and purified water to obtain activated macroporous adsorption resin.
(B) Adsorption: adding the activated macroporous adsorption resin into a biological matrix, mixing and adsorbing, and vacuum filtering to collect the biological matrix. The low-speed mixing adsorption rotating speed is preferably 50-200 rpm, and the mixing adsorption time is 0.5-2 h, preferably 1h.
In an alternative embodiment, the serum albumin comprises Bovine Serum Albumin (BSA).
In an alternative embodiment, the kit further comprises: and (IV) a mobile phase.
In an alternative embodiment, the mobile phase comprises mobile phase a and mobile phase B;
the mobile phase A is an aqueous ammonium formate solution or an aqueous ammonium acetate solution, and contains no more than 0.001% v/v formic acid.
In an alternative embodiment, the concentration of ammonium formate or ammonium acetate in mobile phase a is 2mM.
In an alternative embodiment, the mobile phase A contains 0.001% v/v formic acid.
In an alternative embodiment, mobile phase A is a 2mM ammonium formate aqueous solution containing 0.001% v/v formic acid.
The mobile phase B is methanol solution of ammonium formate or methanol solution of ammonium acetate, and the mobile phase B contains not more than 0.001% v/v formic acid.
In an alternative embodiment, the concentration of ammonium formate or ammonium acetate in mobile phase B is 2mM.
In an alternative embodiment, the mobile phase B contains 0.001% v/v formic acid.
In an alternative embodiment, mobile phase B is 2mM methanolic ammonium formate solution containing 0.001% v/v formic acid.
In a second aspect, there is also provided a method of pre-processing a sample comprising enriching and/or concentrating PCOS-related steroid hormone in said sample using the amphiphilic magnetic solid phase material described in the first aspect, or the kit provided in the first aspect. The samples treated by the pretreatment method can be used for detecting PCOS related steroid hormone by any method acceptable in the field.
Mixing the amphiphilic magnetic solid phase material subjected to activation treatment and balance treatment in sequence with a sample system, enabling the amphiphilic magnetic solid phase material to adsorb PCOS related steroid hormone in a sample, separating the amphiphilic magnetic solid phase material, mixing with a first leaching solution, separating the amphiphilic magnetic solid phase material, mixing with a second leaching solution, eluting with an eluent, and collecting the eluent, wherein the eluent contains the PCOS related steroid hormone in the sample for subsequent detection; the activation treatment comprises mixing an activation solution with the amphiphilic magnetic solid phase material;
The activation treatment comprises mixing an activation solution with the amphiphilic magnetic solid phase material;
the balancing treatment comprises mixing the amphiphilic magnetic solid phase material subjected to the activation treatment with a balancing liquid;
The sample system includes a sample, methanol, and a dissociation liquid.
In an alternative embodiment, the sample system further comprises an internal standard.
In an alternative embodiment, the volume ratio of methanol to dissociation liquid in the sample system is (2-4): 8. Preferably 3:8.
In an alternative embodiment, the mass-to-volume ratio of the amphiphilic magnetic solid phase material to the sample to be tested is 50 (100-500) mg/μl, preferably 50:200 mg/μl.
In the sample pretreatment method, an activating solution, a balancing solution, a dissociation solution, a first eluent, a second eluent and an eluent are composed of the following components:
The activating solution comprises methanol;
The balance liquid comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-10%, and the ratio of the methanol is not 0; the volume ratio of methanol to water may be, for example, but not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably the volume ratio of methanol to water is 5%.
The dissociation solution comprises an ammonium acetate-acetic acid buffer solution with a pH in the range of 5-6, preferably 5.5.
The first leaching solution comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-30%, and the ratio of the methanol is not 0; the volume ratio of methanol to water may be, for example, but not limited to, 1%, 5%, 10%, 15%, 20%, 25% or 30%, preferably the volume ratio of methanol to water is 15%.
The second eluent comprises one or more of n-hexane, isohexane and cyclohexane, preferably n-hexane.
The eluent comprises one or more of methanol, acetonitrile and isopropanol.
In a third aspect, there is also provided a method of detecting PCOS-related steroid hormone, comprising enriching and/or concentrating PCOS-related steroid hormone in a sample using the amphiphilic magnetic solid phase material of the first aspect, and then analyzing the PCOS-related steroid hormone in the sample using liquid chromatography tandem mass spectrometry;
In an alternative embodiment, the enriching and/or concentrating the PCOS-related steroid hormone in the sample using the amphiphilic magnetic solid phase material of the first aspect comprises enriching and/or concentrating the PCOS-related steroid hormone in the sample using the sample pretreatment method of the second aspect.
The liquid chromatography conditions included:
The chromatography column employs a C18 chromatography column, which C18 chromatography column includes any C18 chromatography column known in the art, and also includes chromatography columns further incorporating modifying groups based on C18 chromatography columns, such as T3 chromatography columns.
In an alternative embodiment, the chromatographic column is a ACQUITY UPLC HSS T chromatographic column, preferably 50X 2.1mm 1.8 μm in size.
The mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is an aqueous ammonium formate solution or an aqueous ammonium acetate solution, and contains no more than 0.001% v/v formic acid.
In an alternative embodiment, the concentration of ammonium formate or ammonium acetate in mobile phase a is 2mM.
In an alternative embodiment, the mobile phase A contains 0.001% v/v formic acid.
In an alternative embodiment, mobile phase A is a 2mM ammonium formate aqueous solution containing 0.001% v/v formic acid.
The mobile phase B is methanol solution of ammonium formate or methanol solution of ammonium acetate, and the mobile phase B contains not more than 0.001% v/v formic acid.
In an alternative embodiment, the concentration of ammonium formate or ammonium acetate in mobile phase B is 2mM.
In an alternative embodiment, the mobile phase B contains 0.001% v/v formic acid.
In an alternative embodiment, mobile phase B is 2mM methanolic ammonium formate solution containing 0.001% v/v formic acid.
The elution procedure was as follows:
And at 0-6 min, the volume ratio of the mobile phase A to the mobile phase B is (by 55): 45, to 35:65;
At 6-6.7 min, the volume ratio of the mobile phase A to the mobile phase B is changed from 35:65 to 0:100;
the volume ratio of the mobile phase A to the mobile phase B is kept at 0:100 within 6.7-7.7 min;
And at 7.7-7.9 min, the volume ratio of the mobile phase A to the mobile phase B is changed from 0:100 to 55:45;
and maintaining the volume ratio of the mobile phase A to the mobile phase B at 55 at 7.9-8.5 min: 45.
In an alternative embodiment, the mobile phase flow rate is 0.4 mL/min.
In an alternative embodiment, the column temperature of the chromatographic column is 40 ℃.
In an alternative embodiment, the sample is introduced in an amount of 20. Mu.L.
In alternative embodiments, the mass spectrometry conditions include: ESI+ and ESI-ion ionization positive and negative simultaneous monitoring mode, ion source temperature is 120-150 ℃, capillary voltage is as follows: 1.5-2.5 kV, and solvent gas removal temperature: 400-600 ℃, desolventizing gas: 800-1000L/Hr, taper hole gas: and (3) scanning by adopting multiple reaction monitoring at 0-150L/Hr.
In alternative embodiments, the mass spectrometry conditions include: ESI+ and ESI-ion ionization positive and negative simultaneous monitoring mode, ion source temperature 150 ℃, capillary voltage: 2 kV, desolventizing gas temperature: desolventizing gas at 500 ℃): 1000 L/Hr, taper hole gas: 150 L/Hr, scanning was performed using multiplex reaction monitoring.
In an alternative embodiment, the mass spectral parameters are as follows:
TABLE 1
Wherein IS represents an internal standard.
In an alternative embodiment, the PCOS-related steroid hormone content is calculated by internal standard analysis.
In an optional embodiment, the method for detecting the PCOS-related steroid hormone further includes detecting a calibrator, drawing a calibration curve according to the concentration of the PCOS-related steroid hormone in the calibrator and the detection result of the liquid chromatography-tandem mass spectrometry, and obtaining the concentration or content of each steroid hormone in the sample to be detected by conversion according to the detection result of the liquid chromatography-tandem mass spectrometry of the sample to be detected.
The PCOS-related steroid hormone in the second and third aspects described above comprises one or more of progesterone, 17 a-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, 4-androstenedione, testosterone, 17 a-hydroxy pregnenolone, and dihydrotestosterone; optionally comprising progesterone, 17 alpha-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone sulfate, 4-androstenedione, testosterone, 17 alpha-hydroxy pregnenolone, and dihydrotestosterone.
The samples in the second and third aspects described above include, but are not limited to, samples of blood, body fluid, urine, saliva, tissue or cells isolated from an organism, which samples also optionally include samples containing known levels of PCOS-related steroid hormone for use in constructing a standard curve or for use in validating test results.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1
The embodiment provides a steroid hormone detection kit, comprising:
Amphiphilic (hydrophilic lipophilic) magnetic solid phase materials; preparing a calibrator and a quality control product containing steroid hormones in different concentrations in a biological matrix; an internal standard working solution; also included are solutions used in the sample pretreatment process:
activating solution: pure methanol;
Balancing solution: 5% v/v methanol-water;
dissociation liquid: an ammonium acetate-acetic acid buffer solution having a pH of 5.5;
leacheate 1 (leacheate I): 15% v/v methanol-water;
Leacheate 2 (leacheate ii): n-hexane;
Eluent: pure acetonitrile.
1. The amphiphilic (hydrophilic lipophilic) magnetic solid phase material is prepared by the following steps:
(1) Preparing magnetic particles by a sol-gel method: 1 g of Fe 3O4 was suspended in 90 ml of water and sonicated for 30 minutes. Subsequently, 1.7 g of cetyltrimethylammonium bromide CTAB and 1 g of triethanolamine TEOA were added and the mixture was stirred continuously in a constant temperature water bath at 80℃for 1 hour using a mechanical stirrer. The SiO2@Fe 3O4 recovered by filtration is washed with 50mL of ethanol, taken out and dried in an oven at 60 ℃ for 24 hours, and then calcined at 540 ℃ for 7 hours to obtain SiO 2@Fe3O4 magnetic particles.
(2) One-pot method for preparing magnetic solid phase extraction material: 200mg of humic acid were dissolved in 100 ml of distilled water in a round-bottomed flask, then 2g of SiO2@Fe 3O4 magnetic particles after calcination with air were added, and the suspension was stirred for 2 minutes. The water was removed by rotary evaporator and the resulting solid was subjected to pyrolysis treatment in a stream of N 2 at 600 ℃ in an alumina combustion boat inside a quartz tube for 1 hour to convert into hydrophilic-lipophilic balanced carbonaceous phase magnetic particles. The magnetic particles were washed with a large amount of distilled water in a filter flask until the eluate was neutral.
2. The blank matrix is prepared by the following steps:
Placing 500g of macroporous adsorption resin in a beaker, adding ethanol for soaking, and filtering the solvent by using a Buchner funnel and medical absorbent cotton gauze; and washing with ethanol and purified water for 2 times to obtain activated macroporous adsorption resin. 300g of activated macroporous adsorption resin is added into 900mL of bovine serum, and mixed adsorption is carried out for 1h by using an electric stirrer at a rotating speed of 75rpm, and a blank calf serum matrix is obtained by vacuum filtration and collection.
3. Preparation of calibration material and quality control material
3.1 The formulation scheme of the SW6 intermediate solution is shown in the following table:
TABLE 2 preparation scheme for SW6 intermediate solution
As shown in table 2, a 250mL volumetric flask is taken, the primary stock solution of each analyte is added according to the table, vortex mixing is carried out for 5min, then 50% v/v methanol-water solution is added to the scale mark for constant volume, and the mixture is inverted and mixed for 2min to obtain the intermediate solution of the highest point SW6 of the calibrator.
3.2 The formulation protocol for the calibrator is shown in the following table:
Table 3 preparation scheme of SW1.about.6 intermediate solution
The concentrations indicated above are exemplified by progesterone concentrations, the remaining concentrations being diluted by the same factor. The SW1-SW5 intermediate was diluted with the SW6 intermediate obtained in 3.1 according to the protocol shown in the above table. And then respectively taking 1 volume of intermediate liquid, adding 9 volumes of blank matrix liquid obtained after the treatment in the step2, and fully mixing for 30 min to prepare 6 calibrants J1-J6 with different concentrations. The concentrations of each analyte in the calibrator are shown in the following table:
table 4 concentration of each analyte in the calibrator
3.3 The formulation scheme of the quality control product is shown in the following table:
table 5 formulation scheme for quality control
The concentrations indicated above are exemplified by progesterone concentrations, the remaining concentrations being diluted by the same factor. The intermediate solution of QCL and QCH was obtained by dilution with the intermediate solution of SW6 obtained in 3.1 according to the scheme shown in the above table. And then respectively taking 1 volume of intermediate liquid, adding 9 volumes of blank matrix liquid obtained after the treatment in the step 2, and fully mixing for 30min to prepare 2 quality control products QCL and QCH with different concentrations. The concentrations of each analyte in the quality control are shown in the following table:
TABLE 6 concentration of analytes in quality control substances
4. The preparation of the internal standard working solution is shown in the following table:
table 7 internal standard working solution formulation
And diluting the internal standard stock solution into an internal standard working solution with corresponding concentration according to the table by adopting methanol as a diluent.
Example 2
This example provides a method for detecting steroid hormones using the kit provided in example 1.
1. Sample pretreatment:
1.1 The operation process of the magnetic solid phase extraction pretreatment comprises the following steps:
Taking a deep hole plate for pretreatment process, adding the solvent used in the whole treatment process according to the layout of the solvent and the sample shown in Table 8, wherein 50mg of magnetic adsorption material, 200 mu L of calibrator, quality control product or sample to be tested (blood or serum) are added to position 3, then 50 mu L of internal standard solution is sequentially added to each hole, and vortex mixing is carried out for 3min; then, 150. Mu.L of methanol solution and 400. Mu.L of dissociation solution were added.
From the layout, samples can be added in the number 3 and the number 9, and each plate can be used for preprocessing up to 16 samples.
TABLE 8
And then using an automatic sample processor to sequentially perform magnetic bead activation, magnetic bead balancing, sample dissociation adsorption, first leaching, second leaching and elution according to the mixing mode, mixing time and adsorption time of the following table. The specific steps are as follows:
step (1), taking a deep pore plate, adding a sample to be tested and an internal standard solution into position 3, and uniformly mixing; adding methanol and dissociation liquid;
and (2) activating: adding magnetic beads into the No.1 position, and activating the magnetic beads by using methanol;
step (3) balancing: moving the magnetic beads obtained after the treatment in the step (2) into a No.2 position by using an automatic sample treatment instrument, and balancing the magnetic beads by using a methanol aqueous solution;
And (4) extracting: using an automatic sample processor to move the magnetic beads obtained after the treatment in the step (3) into a number 3 position, and uniformly mixing;
step (5) leaching 1: moving the magnetic beads into the No. 4 eluent 1 by using an automatic sample processor, and uniformly mixing;
step (6) leaching 2: transferring the magnetic beads subjected to the first leaching into No. 5 leaching solution 2 by using an automatic sample treatment instrument, and uniformly mixing;
step (7) elution: using an automatic sample processor to move magnetic beads into the eluent, uniformly mixing, then removing the magnetic beads, diluting the eluent on line, transferring to an upper sample plate, and preparing for on-line test;
TABLE 9
2. Sample detection:
2.1 Chromatographic conditions:
Liquid chromatograph-tandem mass spectrometer: waters Xevo TQS;
The chromatographic column adopts ACQUITY UPLC HSS T chromatographic columns with the specification of 50 multiplied by 2.1mm 1.8 mu m;
mobile phase a was formic acid (0.001% v/v) -2mM ammonium formate-water;
Mobile phase B (aqueous phase) was formic acid (0.001% v/v) -2mM ammonium formate-methanol;
The column temperature was 40.0℃and the sample loading was 20. Mu.L. The liquid phase elution procedure is shown in the following table:
table 10 elution procedure
2.2 Mass spectrometry conditions
Electrospray ionization (ESI) source, ESI+ and ESI-ion ionization were monitored simultaneously in both positive and negative modes (note: DHEAS is ESI-mode). Ion source temperature 150 ℃, capillary voltage CAPILLARY:2 kV, desolventizing air temperature Desolvation Temp:500 ℃, desolventizing gas Desolvation:1000 L/Hr, taper hole gas Cone:150 L/Hr. Scanning was performed using Multiplex Reaction Monitoring (MRM). The information of mass spectrum ion pair parameters and the like are shown in the following table:
table 11 mass spectral parameters
Example 3
Performance verification of steroid detection method provided in example 2:
1. linear range:
The processed calibrator was checked on-machine according to the calibrator formulation method of step 3.2 in example 1. The concentrations of the calibrator are shown in Table 4, and the detection results are shown in FIG. 1-FIG. 26, so that all chromatographic peaks are good in peak type, baseline separation is realized with other analytes, linearity is good, correlation coefficient is over 0.99, and all 13 analytes simultaneously meet recovery rate of 90% -110% and meet quantitative requirements.
2. Precision of
The quality control product was subjected to parallel experiments for 3 times a day for 5 days by using the detection method of example 2, and the average value of the test values of the 3 experiments was taken as the actual measurement value, and the intra-batch and inter-batch variation coefficients and the total variation coefficient of the detection result were examined, with the following results:
Table 12
3. Accuracy:
steroid hormone standard substances with different concentrations are added into blank human serum matrix to prepare samples with low, medium and high concentration levels, the concentrations are respectively shown in the following table, a labeling experiment is carried out, 3 samples are treated in parallel after labeling, and the relative deviation of the sample results after labeling is calculated. The results are shown in the table:
TABLE 13
Example 4
Optimization of cracking temperature of amphiphilic magnetic solid phase extraction material
In this example, silica-coated magnetic particles SiO 2@Fe3O4 were prepared according to the sol-gel method of example 1, step 1. 200 mg of humic acid were then dissolved in 100 ml of distilled water in a round-bottomed flask, then 2g of air calcined SiO 2@Fe3O4 magnetic particles were added and the suspension was stirred for 2 minutes. The water was removed by rotary evaporator, and the obtained solid was subjected to pyrolysis treatment in a stream of N 2 at 300℃and 600℃and 900℃in an alumina combustion boat in a quartz tube, respectively, to convert into hydrophilic-lipophilic balanced carbonaceous phase magnetic particles. The magnetic particles were washed with a large amount of distilled water in a filter flask until the eluate was neutral. To obtain an adsorbent 1 (300 ℃ C. Cracking), an adsorbent 2 (600 ℃ C. Cracking) and an adsorbent 3 (900 ℃ C. Cracking).
The QCH intermediate solution of example 1 was diluted 10-fold with pure water, extracted and detected by the method of example 2 using the above 3 adsorbent materials, and the extraction recovery rate of each analyte was calculated by comparing the peak area with the analyte obtained by direct sample injection detection. The results are shown in the following table:
TABLE 14
As shown in the above table, different cleavage temperatures have a large impact on absolute recovery during extraction. Therefore, the cracking temperature is controlled in the preparation process, so that the magnetic solid phase extraction filler can be better combined with steroid hormone, the extraction efficiency and the quantitative accuracy are improved, and the requirement of clinical high-accuracy detection of the steroid hormone is met. The optimum cleavage temperature is 600 ℃.
Example 5
Mobile phase optimization:
In practical sample testing we found that there was a varying degree of interference between the 17-hydroxy pregnenolone internal standard and the dihydrotestosterone analyte, and that it was difficult to completely separate the impurity peaks using conventional 0.1% v/v formic acid containing mobile phases. The formulations of the mobile phases used and the comparison results are shown in the following table, and the rest of the detection conditions are the same as those of example 2:
TABLE 15
The results are shown in Table 15, and the chromatograms of dihydrotestosterone using group 1 mobile phases are shown in FIG. 27, and it can be seen that there is an interference peak for dihydrotestosterone; the chromatogram of 17-hydroxy pregnenolone-IS IS shown in FIG. 28, and it can be seen that there IS a large interference peak near 17-hydroxy pregnenolone-IS, and FIGS. 27 and 28 illustrate that group 1 mobile phase affects PCOS-related steroid hormone quantification; with group 2 mobile phases, the chromatograms of dihydrotestosterone are shown in fig. 29, and it can be seen that the interference peaks coincide with dihydrotestosterone; the chromatogram of 17-hydroxy pregnenolone-IS IS shown in FIG. 30, and a large interference peak can be seen in 17-hydroxy pregnenolone-IS, and FIG. 29 and FIG. 30 illustrate that group 2 mobile phases affect PCOS-related steroid hormone quantification; with the mobile phase of group 3, the chromatograms of dihydrotestosterone and 17-hydroxy pregnenolone-IS are shown in fig. 31 and 32, respectively, and it can be seen that the interference peak IS completely separated from the target analyte, and fig. 31 and 32 illustrate that the mobile phase of group 3 has the best separation effect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A kit for enriching, concentrating and/or detecting PCOS-related steroid hormones, characterized by comprising an amphiphilic magnetic solid phase material, an activating solution, a balancing solution, a dissociation solution, a first eluent, a second eluent and an eluent;
After mixing humic acid with magnetic particles SiO 2@Fe3O4, carrying out pyrolysis treatment at 300-900 ℃ for 0.5-1.5 h to obtain magnetic particles with surfaces being carbonaceous phases; the magnetic particles SiO 2@Fe3O4 are nano particles with core-shell structures, wherein SiO 2 coats Fe 3O4;
The activating solution comprises methanol;
The balance liquid comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-10%, and the ratio of the methanol is not 0;
The dissociation solution comprises an ammonium acetate-acetic acid buffer solution, and the pH range is 5-6;
the first leaching solution comprises an aqueous solution of methanol, wherein the volume ratio of the methanol to the water is 0-30%, and the ratio of the methanol is not 0;
The second eluent comprises one or more of normal hexane, isohexane and cyclohexane;
the eluent comprises one or more of methanol, acetonitrile and isopropanol;
The PCOS-associated steroid hormone includes one or more of progesterone, 17 alpha-hydroxyprogesterone, 11-deoxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone sulfate, 4-androstenedione, testosterone, 17 alpha-hydroxy pregnenolone, and dihydrotestosterone.
2. The kit of claim 1, wherein the temperature of the pyrolysis treatment is 600 ℃, and the time of the pyrolysis treatment is 1 h;
and/or the mass ratio of the humic acid to the SiO 2@Fe3O4 magnetic particles is 0.05-0.5.
3. The kit according to claim 1, wherein the amphiphilic magnetic solid phase material is prepared according to the following method: dissolving humic acid in water in a reaction vessel, adding SiO 2@Fe3O4 magnetic particles with a formula amount, stirring the suspension, and removing water; and (3) carrying out pyrolysis treatment on the obtained solid in nitrogen flow for 0.5-1.5 h at the temperature of 300-900 ℃, and washing with water after the reaction is finished until the washing waste liquid is neutral.
4. The kit of any one of claims 1-3, further comprising one or more of a calibrator, a quality control, an internal standard, a blank matrix, and a mobile phase;
The blank substrate is a biological substrate comprising an aqueous solution of serum or serum albumin, the biological substrate being pretreated to remove endogenous steroid hormones;
the calibrator and/or the quality control product is prepared from the blank matrix;
the mobile phase comprises a mobile phase A and a mobile phase B;
The mobile phase A is an aqueous ammonium formate solution or an aqueous ammonium acetate solution, and contains no more than 0.001% v/v formic acid;
The mobile phase B is methanol solution of ammonium formate or methanol solution of ammonium acetate, and the mobile phase B contains not more than 0.001% v/v formic acid.
5. The kit of claim 4, wherein the pretreatment for removal of endogenous steroid hormones comprises removal of endogenous steroid hormones from the biological matrix using macroporous adsorbent resin.
6. A sample pretreatment method comprising enriching and/or concentrating PCOS-related steroid hormone in a sample using the amphiphilic magnetic solid phase material according to any one of claims 1 to 5 or using the kit for enriching, concentrating and/or detecting PCOS-related steroid hormone according to any one of claims 1 to 5;
Mixing the amphiphilic magnetic solid phase material subjected to activation treatment and balance treatment in sequence with a sample system, enabling the amphiphilic magnetic solid phase material to adsorb PCOS related steroid hormone in a sample, separating the amphiphilic magnetic solid phase material, mixing with a first leaching solution, separating the amphiphilic magnetic solid phase material, mixing with a second leaching solution, eluting with an eluent, and collecting the eluent, wherein the eluent contains the PCOS related steroid hormone in the sample for subsequent detection;
The activation treatment comprises mixing an activation solution with the amphiphilic magnetic solid phase material;
the balancing treatment comprises mixing the amphiphilic magnetic solid phase material subjected to the activation treatment with a balancing liquid;
the sample system comprises a sample, methanol and dissociation liquid;
The activating solution is the activating solution in claim 1; the balancing liquid is the balancing liquid in claim 1; the dissociation liquid is the dissociation liquid described in claim 1; the first eluent is the first eluent described in claim 1; the second eluent is the second eluent described in claim 1; and, the eluent is the eluent as claimed in claim 1.
7. A method for detecting PCOS-related steroid hormone, comprising enriching and/or concentrating PCOS-related steroid hormone in a sample using the amphiphilic magnetic solid phase material according to claim 1, and then analyzing the PCOS-related steroid hormone in the sample by liquid chromatography tandem mass spectrometry;
The liquid chromatography conditions included:
the chromatographic column adopts a C18 chromatographic column;
the mobile phase system comprises a mobile phase A and a mobile phase B;
The mobile phase A is an aqueous ammonium formate solution or an aqueous ammonium acetate solution, and contains no more than 0.001% v/v formic acid; the mobile phase B is methanol solution of ammonium formate or methanol solution of ammonium acetate, and the mobile phase B contains not more than 0.001% v/v formic acid.
8. The method according to claim 7, wherein the sample pretreatment method according to claim 6 is used for pretreatment.
9. The method according to claim 7, wherein the liquid chromatography is performed by gradient elution, and the elution procedure is as follows:
And at 0-6 min, the volume ratio of the mobile phase A to the mobile phase B is (by 55): 45, to 35:65;
At 6-6.7 min, the volume ratio of the mobile phase A to the mobile phase B is changed from 35:65 to 0:100;
the volume ratio of the mobile phase A to the mobile phase B is kept at 0:100 within 6.7-7.7 min;
And at 7.7-7.9 min, the volume ratio of the mobile phase A to the mobile phase B is changed from 0:100 to 55:45;
and maintaining the volume ratio of the mobile phase A to the mobile phase B at 55 at 7.9-8.5 min: 45.
10. The method of claim 7, wherein the mass spectrometry conditions comprise: ESI+ and ESI-ion ionization positive and negative simultaneous monitoring mode, ion source temperature is 120-150 ℃, capillary voltage is as follows: 1.5-2.5 kV, and solvent gas removal temperature: 400-600 ℃, desolventizing gas: 800-1000L/Hr, taper hole gas: and (3) scanning by adopting multiple reaction monitoring at 0-150L/Hr.
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