1. A method for simultaneously detecting multiple exogenous and endogenous steroid hormones, comprising the steps of:

A. pretreating a sample to be detected;

B. b, loading the sample to be detected processed in the step A on an SPE plate;

C. adding a mixed solution of acetonitrile and water for elution;

D. adding hexane for elution, and discarding the eluent;

E. adding a mixed solution of methanol and acetonitrile for elution, and collecting eluent;

F. e, diluting the eluent in the step E, injecting the diluted eluent into an LC-MS mass spectrometer for detection and analysis to obtain the content of a plurality of exogenous and endogenous steroid hormones;

the detection types of the exogenous steroid hormones and the endogenous steroid hormones are 54 or more.

2. The method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 1, wherein in the step A, the sample to be detected is a serum sample or a urine sample.

3. The method according to claim 2, wherein the pretreatment method comprises the following steps when the sample to be tested is a serum sample: adding methanol and water into the serum sample, carrying out vortex centrifugation, and carrying out subsequent treatment on the obtained supernatant.

4. The method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 2, wherein when the sample to be detected is a urine sample, the pretreatment method comprises the following steps: adding phosphate buffer solution into the urine sample, adding glucuronyl acidifying enzyme for enzymolysis, incubating at 55 ℃ for 1 hour, and cooling to room temperature; the resulting supernatant is subjected to subsequent treatment.

5. The method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 4, wherein the volume ratio of the added urine sample, phosphate buffer and glucuronidase is 2:1: 0.05.

6. the method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 1, wherein in the step C, the volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water is 1: 9;

in the step D, the hexane is n-hexane;

in the step E, the volume ratio of the methanol to the acetonitrile in the mixed solution of the methanol and the acetonitrile is 1: 9.

7. The method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 1, wherein in step F, the LC-MS mass spectrometer is used for analysis under the following liquid phase analysis conditions:

the chromatographic column adopts an ACQUITY BEH C18 chromatographic column;

the adopted mobile phase A is water; the mobile phase B is a mixed solution of acetonitrile and an ammonium formate aqueous solution containing 0.1 percent of FA, wherein the concentration of the ammonium formate aqueous solution in the mixed solution is 10 mM;

the mobile phase gradient conditions used were:

time (min) Flow rate (mL/min) Phase A Phase B 0 0.3 50 50 3 0.3 50 50 7 0.3 0 100 7.2 0.4 0 100 14.0 0.4 0 100 14.2 0.3 0 100 15.0 0.3 50 50

。

8. The method for simultaneously detecting multiple exogenous and endogenous steroid hormones according to claim 1, wherein when the exogenous and endogenous steroid hormones to be simultaneously detected are 54 compounds, the mass spectrometric conditions used are:

Background

The measurement of steroid hormones in biological samples is reported in published papers, but most of the measurement is of steroid hormones in vivo, also called as measurement of steroid hormones, and the measurement of exogenous steroid hormones in vivo has few reports, and no report is available for simultaneously measuring exogenous and endogenous 54 steroid hormones.

In the prior literature and published patents, there are some methods for measuring steroid hormones, but because of the numerous types and similar structures of steroid hormones, there are still great challenges in analysis. The main challenges include: the steroid hormones are various in types and can be divided into endogenous steroid hormones and exogenous steroid hormones from the source, the endogenous steroid hormones are derived from the self secretion of a human body or an animal body, are important chemical components for normal operation of body functions, are used as one of the hormones and play an important role in regulating the biological functions of the human body and the animal body; exogenous steroid hormones mainly come from medicines (including human medicines and veterinary medicines, a large amount of steroid hormones are used in medicines, particularly glucocorticoids, and the exogenous steroid hormones in human bodies are increased due to improper use of part of medicines), foods (mainly coming from residues of veterinary medicines, and steroid hormones in veterinary medicines can remain in foods of animal origin and are taken by human bodies through food routes, including animal muscles, livers, kidneys, milk products and other related meat and milk products; in addition, illegal addition of steroid hormones in health-care foods can also become one of the sources exposed to steroid hormones in human bodies), environments (mainly entering the environments through human and animal excretions, discharge of wastewater of water treatment plants and pharmaceutical factories and the like, and sewage, animal farms and aquatic farms are the main sources of steroid hormones in the environments; the current sewage treatment systems cannot completely remove the steroid hormones, so that steroid hormone and metabolite thereof are accumulated in the environment and are further absorbed into the human body through the environment), cosmetics (glucocorticoid is the most common externally-applied medicine in dermatology, can inhibit the regeneration of fiber cells, has certain whitening effect on the skin, but can generate hormone dependence and other side effects after long-term use, even cause cancer and cause serious injury to the human body. In China, the 'cosmetic hygiene Specification' 2007 edition clearly stipulates that glucocorticoids belong to forbidden substances. But many manufacturers still illegally add glucocorticoid substances to cosmetics due to obvious whitening, acne removing and other effects), the list of exogenous steroid hormones and endogenous steroid hormones is shown in the following table:

the exogenous steroid hormone and the endogenous hormone have certain similarity in structure, and the exogenous hormone is a non-in-vivo self-secreted hormone and is derived from an artificially synthesized hormone. Exogenous hormones, which are also called endocrine disruptors because they have a great influence on the human body, interfere with the normal functions of the endocrine system of the organism at low concentrations, and long-term exposure to the endocrine disruptors at low concentrations may cause a series of reproductive and developmental problems. It has been found that a decline in the energy of men, an increase in the incidence of breast cancer in women, and a decline in immunity in newborns are all associated with endocrine disruptors. Animal survey shows that some birds have thinned eggshells, reduced hatchability, reduced seal immunity, obvious fish feminization and degenerated male genital glands. Although the influence of steroid hormones on the health condition of organisms is great at present, reports on the in vivo quantitative determination of the steroid hormones are very limited, and only few exogenous steroid hormones are concentrated on the reports occasionally, and all the common exogenous steroid hormones on the market are not covered. In addition, it has been reported that only endogenous steroid hormones in vivo are measured. The amount of steroid hormones in the body is often used to assess the pathological state of a human or organism, such as 21-hydroxylase deficiency, 11 beta-hydroxylase deficiency, 17-hydroxylase deficiency, 3-hydroxysteroid dehydrogenase deficiency, polycystic ovary syndrome, and the like. However, the absolute quantification of all steroid hormones in the body, particularly the absolute quantification of the content of exogenous steroid hormones in the body, is of great importance for the evaluation of the health status of a human body. Because exogenous steroid hormones have great influence on human bodies, long-term exposure causes a plurality of potential health problems, and the compounds are serious potential risks for diseases, can be detected as early as possible, and are vital to the health of human bodies.

However, the current methods for measuring endogenous and exogenous steroid hormones in human and animal bodies are very limited, and particularly, the simultaneous measurement of all major exogenous steroid hormones and endogenous steroid hormones has not been reported.

Simultaneous determination of numerous exogenous and endogenous hormones in vivo presents the following challenges: (1) the steroid hormones have similar structures and even a plurality of isomers, particularly when a large number of steroid hormones are simultaneously measured together, the problem is more prominent, and the measurement of only 20-30 steroid hormones is very challenging, but with the development of economy and market, the measurement of only 20-30 hormones is far from enough, so that the problems of good separation and accurate measurement of the hormones must be solved; (2) the content of steroid hormones in vivo is greatly different from each other, some hormones are very high, some hormones are very low, when a plurality of hormones are simultaneously measured, high-concentration hormones and low-concentration hormones need to be considered, and the biggest challenge in solving the problem lies in the optimization of the pretreatment condition of a sample and the setting of the condition of a detector; (3) the detection of hormones in vivo is complicated by using a biological sample matrix, and when the number of hormones to be analyzed is more and more, the hormones and the matrix can be separated as much as possible to reduce the influence of the matrix; (4) the steroid hormone exists mainly in the form of prototypes in blood, mainly exists in the form of glucuronic acid conjugates and prototypes in urine, needs special treatment, and cannot directly express the exposure of the steroid hormone in vivo according to the content of the prototypes of the steroid hormone in urine, so that the measurement result is low, and the analysis result and the evaluation scheme are unreasonable; (5) although the number of steroid hormones to be quantified simultaneously is large, the analysis time is as short as possible, because in vivo samples are usually many, the pretreatment time and the analysis time of the samples must be simplified as much as possible in the case of analysis of thousands of samples, otherwise, the requirements of large sample detection amount and rapid report of in vivo samples cannot be met.

Among them, patent document CN103698414A describes a method for detecting steroid hormones in urine based on chemical derivatization, which provides a method for detecting steroid hormones in urine after derivatization by using 4-dimethylaminobenzoic acid as a novel derivatization reagent and combining a high-resolution and high-sensitivity liquid chromatography-mass spectrometry detection instrument. However, the derivatization in this patent takes too long, and the derivatization reaction alone requires 5 hours in a 75 ℃ water bath. For clinical samples, derivatization alone takes 5 hours without counting other operations, which may be really too long, and clinical sample detection has no way to wait for this long time. In addition, the bath temperature of 75 ℃ is too high, which has a great influence on the stability of the hormone, generally speaking, the hormone standard and the biological sample are preserved at-20 ℃ or-80 ℃ to ensure the stability, and under the condition of long-time bath with the high temperature, the content of a plurality of hormones can be changed and cannot be accurately measured. In addition, the patent only detects the endogenous steroid hormones and the time for analyzing a sample in a mass spectrometer is more than 37 minutes (see patent drawings, the last chromatographic peak is 37 minutes, and the time for regenerating a chromatographic column is not shown in the drawings), and the time for analyzing a clinical sample is also too long when the time is more than 37 minutes. Together with sample extraction, derivatization reactions and mass spectrometry, the analysis period for one sample takes approximately 6-7 hours. The time reported for each sample was too long and could not be used for large-scale population measurements.

Patent document CN110187043A describes a method for simultaneously detecting 13 steroid hormones in serum, in which the detection solution is detected by using the hplc-tandem mass spectrometry technique, and only the endogenous steroid hormones in blood are detected, and the number of the steroid hormones to be detected is limited, and is limited to only the endogenous steroid hormones, and is limited to only the blood sample. Patent document CN 106198786 a describes a method for rapidly detecting 5 steroid hormones in human urine by using UPLC-MS/MS technology, which combines the enzymatic hydrolysis of β -glucuronidase with UPLC-MS/MS technology. The steroid hormones detected are very limited and limited to endogenous steroid hormones, the enzymolysis time is 3 hours, and the requirement of high-throughput analysis on clinical samples is not met.

Disclosure of Invention

In view of the deficiencies in the prior art, it is an object of the present invention to provide a method for simultaneously detecting a plurality of exogenous and endogenous steroid hormones.

The purpose of the invention is realized by the following technical scheme:

the invention provides a method for simultaneously detecting a plurality of exogenous and endogenous steroid hormones, which comprises the following steps:

A. pretreating a sample to be detected;

B. b, loading the sample to be detected processed in the step A on an SPE plate;

C. adding a mixed solution of acetonitrile and water for elution;

D. adding hexane for elution, and discarding the eluent;

E. adding a mixed solution of methanol and acetonitrile for elution, and collecting eluent;

F. d, diluting the eluent in the step D, injecting the diluted eluent into an LC-MS mass spectrometer for detection and analysis to obtain the content of a plurality of exogenous and endogenous steroid hormones;

the detection types of the exogenous steroid hormones and the endogenous steroid hormones are 54 or more.

Preferably, in step a, the sample to be tested is a serum sample or a urine sample.

Preferably, when the sample to be detected is a serum sample, the pretreatment method comprises: adding methanol and water into the serum sample, carrying out vortex centrifugation, and carrying out subsequent treatment on the obtained supernatant.

Preferably, when the sample to be detected is a urine sample, the pretreatment method comprises the following steps: adding phosphate buffer solution into the urine sample, adding glucuronyl acidifying enzyme for enzymolysis, incubating at 55 ℃ for 1 hour, and cooling to room temperature; the resulting supernatant is subjected to subsequent treatment.

Preferably, the volume ratio of the urine sample, the phosphate buffer solution and the glucuronidase is 2:1: 0.05; if too much phosphate buffer (for example, 2:2:0.05) or too little (for example, 2:0.5:0.05), too much glucuronidase (for example, 2:1:0.1) or too little (for example, 2:1:0.02) is used, the release rate of steroid hormone to be detected in urine and the stability of the experiment are affected, the amount of glucuronidase does not need to be increased any more, and the continuous increase is waste of enzyme. The adding volume ratio of the urine sample to the phosphate buffer solution to the glucuronyl acidifying enzyme is 2:1:0.05, the optimal formula for enzymolysis is provided, the optimal effect and the optimal time can be achieved, stable and efficient enzymolysis release can be completed within 1 hour, the pretreatment time of the sample is greatly reduced, and the optimal effect and efficiency are achieved.

Preferably, the glucuronidase is beta-glucuronidase from Roman snail. The beta-glucuronidase is of various types and respectively comes from escherichia coli, bovine liver and calophyllum (Patella vulgaris vulgata), and different beta-glucuronidases are found to have different effects on the release of the steroid hormone conjugate through experiments. The beta-glucuronidase from the roman snail is suitable, can stably release all steroid hormones to be detected in urine, does not interfere the stability of the steroid hormones, and cannot fully release the steroid hormones from other beta-glucuronidases such as the escherichia coli beta-glucuronidase.

Preferably, in the step C, the volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water is 1: 9; the ratio of the elution solution for removing impurities and the matrix is optimized, the ratio of acetonitrile to water is the optimal ratio when the ratio is 1:9, the retention of steroid hormones on the SPE plate is not influenced, and the impurities and the background matrix can be removed to the maximum extent.

In the step D, the hexane is n-hexane; n-hexane is the optimal elution solution, does not influence the retention of steroid hormones on SPE plates, and can remove impurities with weak polarity and background matrix to the maximum extent.

In the step E, the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 1: 9; the optimal solution for eluting steroid hormone is optimized, and the ratio of methanol to acetonitrile is 1:9, so that the steroid hormone can be sufficiently eluted from the SPE plate.

Preferably, in step E, when the LC-MS mass spectrometer is used for analysis, the liquid phase analysis conditions are as follows:

the chromatographic column adopts an ACQUITY BEH C18 chromatographic column;

the adopted mobile phase A is water; the mobile phase B is a mixed solution of acetonitrile and an ammonium formate aqueous solution containing 0.1 percent of FA, wherein the concentration of the ammonium formate aqueous solution in the mixed solution is 10 mM;

we have found that good separation of such a large amount of steroid hormones cannot be achieved with only 0.1% FA (formic acid), ammonium formate is a very effective modifier and ammonium acetate is less effective than ammonium formate. In addition, the concentration of ammonium formate also has an influence on the separation effect, 10mM ammonium formate can achieve the best separation effect, excessive ammonium formate concentration (more than 20mM, including 20mM,30mM and 40mM) has certain influence on the response of the compound, and lower ammonium formate concentration (5mM) has insufficient stability on the separation effect of the mobile phase system.

The mobile phase gradient conditions used were:

the gradient of the mobile phase also plays a key role for simultaneous analysis of so many steroid hormones. The same flow rate, such as 0.3ml/min or 0.4ml/min, is always used, and no satisfactory effect can be achieved; within 7 minutes, a flow rate of 0.3ml/min and within 7.2-14 minutes a flow rate of 0.4ml/min was used, enabling good separation of all steroid hormones within a limited analysis period.

Preferably, when the exogenous and endogenous steroid hormones to be detected are 54 compounds, the analysis conditions need to be greatly optimized because of the large number and large content difference of the steroid hormones to be detected simultaneously and the influence of the background matrix.

In the process of optimizing the mass spectrum condition, different parent ions and daughter ions for detection are selected and optimized, different collision voltages and declustering voltages are selected and optimized,

the optimal mass spectrometry conditions are as follows:

all of the parent ions, daughter ions and corresponding mass spectrometry parameters for detection are not optimal except for the final mass spectrometry detection conditions selected. In the selection of the parent ions and the daughter ions, the applicability of different parent ions and daughter ions to analysis is considered, and experiments prove that some parent ions and daughter ions are not suitable for the detection of biological samples although the parent ions and daughter ions are indeed helpful for improving the detection sensitivity, and some ions have the interference of a matrix and are unstable, so that the requirements of the analysis cannot be met.

The main parent ions, daughter ions and corresponding mass spectrum parameters that have been optimized are listed below (experimentally verified that the other mass spectrum parameter conditions shown in the table cannot meet the analysis requirements except for the series of parameters used in the present invention):

compared with the prior art, the invention has the following beneficial effects:

1. the method of the invention realizes the simultaneous detection of at least 54 steroid hormones in the same sample, wherein the 54 steroid hormones cover all the common exogenous steroid hormones (42) and the main endogenous hormones (13) which are from medicines, foods, environments and the like and expose human bodies and animal bodies. By this method, the exogenous hormone exposure level and the endogenous hormone level of an organism can be rapidly evaluated. The method is simple and convenient to operate, only needs 15 minutes for analysis, establishes and verifies 2 most main biological matrixes (serum and urine), and can be used for accurately measuring the content of steroid hormones in the serum and the urine in the future. The invention greatly improves the speed and flux of the detection of the steroid hormones in vivo and the types and the quantity of the covered steroid hormones, and can carry out rapid and comprehensive evaluation on various organisms, patients and healthy people. The characteristics directly related to the steroid hormones can be rapidly analyzed and evaluated by the method.

2. The invention provides an analysis method especially aiming at the high risk hidden trouble caused by the exposure of a plurality of exogenous steroid hormones from medicines, foods, environments and cosmetics, so that the detection and the intervention can be carried out as early as possible, and the potential disease risk in the future is avoided.

3. According to the invention, the serum samples and the urine samples of 47 children are measured, and the results show that the content change trends of steroid hormones in the serum samples and the urine samples are consistent, and the results show that the urine can replace the serum to be used as a biological sample for evaluation of the steroid hormones.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a chromatogram of 54 steroid hormones analyzed in example 1.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1 determination of limit of quantitation, Standard Curve, regression equation and chromatogram for 54 steroid hormones

Step one, respectively preparing standard substance solutions with different concentrations of 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 10ng/ml and 100ng/ml for 54 standard substances of steroid hormones;

step two, respectively taking 250 microliters of the standard solutions of the 54 steroid hormones, adding 250 microliters of methanol, adding 250 microliters of water, carrying out vortex centrifugation, and precipitating protein;

taking 600 microliters of supernatant, and sampling on an SPE plate;

step four, adding acetonitrile: eluting with water (1: 9);

step five, adding 200 microliters of hexane for elution, and discarding the eluent;

step six, adding 30 microliters of methanol/acetonitrile (1:9), and collecting the eluent;

step seven, adding 30 microliters of water into the eluent for dilution;

step eight, injecting the diluent (10 microliters) into an LC-MS mass spectrometer for analysis;

the chromatographic column adopts an ACQUITY BEH C18 chromatographic column;

the adopted mobile phase A is water; the mobile phase B is a mixed solution of acetonitrile and an ammonium formate aqueous solution containing 0.1 percent of FA, wherein the concentration of the ammonium formate aqueous solution in the mixed solution is 10 Mm;

the mobile phase gradient conditions used are shown in table 1:

TABLE 1

The specific mass spectrometry conditions employed are shown in table 2:

TABLE 2

The standard regression equation and linear regression coefficient of 54 steroid hormones obtained by analyzing the standard solutions with different concentrations of the 54 steroid hormone series are shown in table 3.

TABLE 354 Standard regression equation and regression coefficients for steroid hormones

NA: the detection limit is the highest point of the standard curve, and the content is calculated by adopting a single point;

the lowest point of the standard curve is taken as the detection quantitative limit LOQ of 54 steroid hormones, and the detection quantitative limit of 54 steroid hormones is shown in the following table 4.

TABLE 4

The chromatogram results for 54 steroid hormones are shown in fig. 1, which shows a superimposed graph of the MRM total ion flow graph for 54 steroid hormones in fig. 1.

Example 2 method recovery of simultaneous determination of 54 steroid hormones

Step one, taking 250 microliters of 54 steroid hormone mixed standard substance solution (10ng/mL), adding 250 microliters of methanol, adding 250 microliters of water, carrying out vortex centrifugation, and precipitating protein;

taking 600 microliters of supernatant, and sampling on an SPE plate;

step three, adding acetonitrile: eluting with water (1: 9);

step four, adding 200 microliters of hexane for elution, and discarding the eluent;

step five, adding 30 microliters of methanol/acetonitrile (1:9), collecting eluent, and adding 30 microliters of water into the eluent for dilution;

injecting the diluent (10 microliters) into an LC-MS mass spectrometer for analysis;

and step seven, comparing the peak area of the compound with the peak area of the unextracted standard substance to obtain the recovery rate.

The specific liquid phase analysis conditions and mass spectrometry conditions used were the same as in example 1.

The results of the method recovery calculations are shown in Table 5.

Table 554 steroid hormone method recovery calculations

NA: the concentration for recovery rate investigation did not reach the detection limit of the compound.

EXAMPLE 346 determination of the content of 54 steroid hormones in the serum of children

The embodiment provides a method for measuring the content of 54 steroid hormones, which comprises the following steps:

the method comprises the following steps: collecting serum sample according to standard method, storing at-80 deg.C until the sample is ready for determination;

step two: taking a 250 microliter serum sample, adding 250 microliter methanol, adding 250 microliter water, carrying out vortex centrifugation, and precipitating protein;

step three: taking 600 microliter of supernatant, and loading on an SPE plate (SPE plate type number: Oasis PRIME HLB mu Elution 96 plate);

step four: adding mixed solution of acetonitrile/water (volume ratio is 1:9) for elution, then adding 200 microliter of hexane for elution, and discarding the eluent;

step five: adding 30 microliters of mixed solution of methanol/acetonitrile (volume ratio is 1:9), collecting eluent, and adding 30 microliters of water into the eluent for dilution;

step six: the dilution (10. mu.l) was injected into an LC-MS mass spectrometer for analysis; the specific liquid phase analysis conditions and mass spectrometry conditions used were the same as in example 1.

54 steroid hormones in the serum of 46 children were measured by the method of the present invention, and the results are shown in Table 6. Wherein:

cases sexual precocity group (n ═ 23)

Control: health children group (n ═ 23)

Average concentration of Serum of children in sexual precocity group (ng/mL) (n ═ 23)

SD of plasma in cases (n ═ 23): bias of individual steroid hormones (ng/mL) in the serum of children in precocious puberty group (n ═ 23)

Average control of Serum in control (ng/mL) (n 23): average concentration (ng/mL) of individual steroid hormones in serum of children in healthy children group (n ═ 23)

SD of plasma in control (n ═ 23): deviation of individual steroid hormones (ng/mL) in serum of healthy children (n ═ 23)

P values: the T test result of the difference of the contents of the steroid hormones of the precocious group and the healthy child group;

fold Change: the change ratio of the steroid hormone content of the precocious group and the healthy children group.

TABLE 6

NA: not detected out

The results of 54 steroid hormones in the serum of 3 individual premature children (samples 1-3) and 3 healthy children (samples 4-6) measured therein are shown in table 7.

TABLE 7

NA: not detected out

EXAMPLE 346 determination of the content of 54 steroid hormones in urine of children

The embodiment provides a method for measuring the content of 54 steroid hormones, which comprises the following steps:

the method comprises the following steps: collecting urine sample according to standard method, storing at-80 deg.C until the sample is ready for determination;

step two: taking 0.5 ml urine sample, adding 0.25 ml phosphate buffer solution, adding 12.5 microliters of glucuronyl acidifying enzyme derived from the Roman snail for enzymolysis, incubating at 55 ℃ for 1 hour, and cooling to room temperature;

step three: taking 600 microliter of supernatant, and loading on an SPE plate (SPE plate type number: Oasis PRIME HLB mu Elution 96 plate);

step four: adding mixed solution of acetonitrile/water (volume ratio is 1:9) for elution, then adding 200 microliter of hexane for elution, and discarding the eluent;

step five: adding 30 microliters of mixed solution of methanol/acetonitrile (volume ratio is 1:9), collecting eluent, and adding 30 microliters of water into the eluent for dilution;

step six: the dilution (10. mu.l) was injected into an LC-MS mass spectrometer for analysis;

the specific liquid phase analysis conditions and mass spectrometry conditions used were the same as in example 1.

54 steroid hormones in the urine of 46 children were measured by the method of the present invention, and the results are shown in Table 8. Wherein:

cases sexual precocity group (n ═ 23)

Control: health children group (n ═ 23)

Average concentration of Serum in cases (ng/mL) (n ═ 23), Average concentration of individual steroid hormones in urine (ng/mL) of children in precocious puberty group (n ═ 23)

SD of plasma in cases (n ═ 23): deviation (ng/mL) of each steroid hormone in urine of children in precocious puberty group (n ═ 23)

Average control of Serum in control (ng/mL) (n 23): average concentration of each steroid hormone in urine (ng/mL) for children in healthy children group (n ═ 23)

SD of plasma in control (n ═ 23): deviation of each steroid hormone (ng/mL) in urine of healthy children (n ═ 23)

P: t test result of difference of steroid hormone content between precocious puberty group and healthy child group

Fold Change: the change ratio of the steroid hormone content of the precocious group and the healthy children group.

TABLE 8

NA: not detected out

The results of 54 steroid hormones in the sera of 3 individual premature children (samples 1-3) and 3 healthy children (samples 4-6) which were the same as in example 3 were determined as shown in table 9.

TABLE 9

NA: not detected out

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.