Method for simultaneous detection of multiple biomarkers of precocious puberty

Abstract

The present invention discloses a method for simultaneously detecting multiple precocious puberty biomarkers; the method comprises the following steps: 1) taking a urine sample and adding an internal standard solution; 2) enzymolysis: adding a glucuronidase solution to the sample solution of step 1) for enzymolysis; 3) the samples after enzymolysis are respectively loaded on an HLB solid phase extraction plate and a MAX solid phase extraction plate, and after washing with a rinsing liquid to remove interfering substances, the samples are eluted with an eluent, and the eluents of the HLB solid phase extraction plate and the MAX solid phase extraction plate are combined, and UPLC-MS detection is performed to obtain the contents of multiple precocious puberty biomarkers; the multiple precocious puberty biomarkers include exogenous hormone compounds, plasticizers, bisphenol A compounds, and endogenous hormone compounds; the detection types of the multiple precocious puberty biomarkers are 102 or more. The present invention is easy to operate and greatly improves the detection speed, throughput, and types and quantity of precocious puberty biomarkers covered.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis technology, and specifically to a method for simultaneously detecting multiple precocious puberty biomarkers, and in particular to a method for simultaneously detecting exogenous hormones, plasticizers, bisphenol A and endogenous hormones. Background Art

[0002] Precocious puberty (PP) is a developmental abnormality characterized by the early onset of sexual maturity. Precocious puberty seriously affects the normal growth and development of children. Precocious puberty children are often accompanied by advanced bone growth, which will cause the child to be short in adulthood. In addition, precocious puberty will also affect the child's psychology and behavior. Precocious puberty children are more likely to feel negative emotions such as inferiority and loneliness, and have poor social adaptability. Precocious puberty in children can be intervened through a variety of means and methods, such as controlling diet, increasing exercise, and giving drug treatment, but all interventions must be implemented in the early stage, and clinical diagnosis can only obtain the results of precocious puberty in children, such as bone age, hormone levels, secondary sexual characteristics, etc., so it is very passive and cannot intervene in precocious puberty in a timely manner.

[0003] Precocious puberty brings such huge harm, which will have a great impact on both families and children, but there is still little we can do about early contact and screening for precocious puberty. At present, in clinical practice, children and their parents usually go to the hospital for medical treatment only when the child has been found to have secondary sexual characteristics. For example, breast development and even menstruation have been found in girls, and secondary sexual characteristics or nocturnal emission have been found in boys. When parents find that their children have shown preliminary secondary sexual characteristics, they will realize that the child has undergone precocious puberty. However, if clinical symptoms have already occurred, then when a bone age scan is performed, it will be found that the sutures have begun to close, which means that the child’s height is likely to stop growing.

[0004] In fact, there are many clinical means and methods to prevent children from precocious puberty, such as asking children to pay attention to their diet, asking children to strengthen physical exercise, and giving specific drugs to prevent children from developing. However, all interventions must be implemented in the early stage. When the sutures have already closed clinically, these treatments will have little effect, or even no way to prevent the development of precocious puberty. The key contradiction now is that there is no effective screening method to detect children's precocious puberty in the clinic. Clinically, the results can be tested, such as bone age, and sex hormones in children, but all of these are the results of precocious puberty, not the cause. There is no way to find the cause of precocious puberty from the source and intervene in a timely and effective manner. Therefore, how to detect the trend and cause of precocious puberty in children as early as possible and provide effective intervention when children have not yet developed secondary sexual characteristics and have not yet developed has become the most urgent clinical problem.

[0005] In order to effectively find the trend and cause of precocious puberty in children, it is necessary to screen the biomarkers that can cause precocious puberty efficiently. However, according to previous studies, the causes of precocious puberty are very complex and diverse. In order to establish a screening method for biomarkers related to precocious puberty as soon as possible, we studied and found and summarized all biomarkers that may be related to precocious puberty. The following is the information of these biomarkers related to precocious puberty.

[0006] 1) Sometimes, in order to make meat-eating animals such as chickens, pigs, and cattle grow as quickly as possible, businesses will add drugs and additives that promote development to the feed. These compounds that promote development will leave residues in the muscles of these animals, and children will indirectly ingest compounds that promote development after eating these meats. These come from various hormones contained in food, cosmetics, feed additives, and medicines, such as alclomethasone dipropionate, amcinonide, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone acetate, betamethasone dipropionate, betamethasone valerate, clobetasone butyrate, budesonide, deflazacort, dexamethasone acetate, diflorasone diacetate, fluorometholone, fluorometholone acetate, flumethasone, fluocinolone acetate, fludrocortisone acetate, flurandrenolide, fluticasone propionate, halcinonide, methylprednisolone, methylprednisolone acetate, cortisone betamethasone propionate, clobetasol propionate, mometasone, prednicarbate, prednisone, prednisone acetate, prednisolone, prednisolone acetate, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, triamcinolone acetonide, triamcinolone acetonide acetate, triamcinolone, triamcinolone diacetate, testosterone propionate, methyltestosterone, betamethasone propionate, betamethasone butyrate propionate, chlormadinone acetate, chlormadinone acetate, ciclesonide, clopredniol, desoxonide, desoximetasone, diethylstilbestrol, diflucortolone valerate, diflugestrol butyl, ethinyl estradiol, flunisolide, flurazepam Causes of precocious puberty in children include: pine, fluocortolone, halobetasol propionate, halometasone, hexestrol, hydrocortisone acetate propionate, hydroxyprogesterone caproate, norgestrel, loteprednol, medroxyprogesterone, medroxyprogesterone acetate, megestrol, megestrol acetate, methandrone, methylprednisolone acetate propionate, mifepristone, mometasone furoate, nandrolone, nandrolone phenylpropionate, norethisterone, norethisterone acetate, paramethasone, paramethasone acetate, isofluprednisolone, isofluprednisolone acetate, chlorethoxyquin, nandrolone decanoate, oxymetholone / anadrol, stanozolol, raloxifene, etc. The causes of precocious puberty in children include Their endocrine disrupting effects, these compounds can mimic or interfere with natural hormones in the human body, especially sex hormones such as estrogen and androgen, interfering with normal hormone signaling, which may lead to an imbalance in hormone levels and affect the sexual maturation process of children; adversely affect the development of the reproductive system of children, such as causing early changes in sex hormone levels, thereby promoting the occurrence of precocious puberty; affect brain development and behavior, especially the function of brain areas related to sexual maturation; and long-term exposure and cumulative effects, children are more sensitive to chemicals in the environment due to their growth and development characteristics. Long-term exposure to environments containing these hormones, even at low doses, may lead to the cumulative effects of health problems, including an increased risk of precocious puberty. The characteristics of growth and development are more sensitive to chemicals in the environment. Long-term exposure to environments containing these hormones, even at low doses, may lead to the cumulative effects of health problems, including an increased risk of precocious puberty.Therefore, regulatory agencies and health organizations usually strictly monitor and restrict the use of these compounds in food, cosmetics, feed additives, and medicines to protect children from these potential health risks. However, it is difficult to know how much hormones children actually ingest from the environment without measuring the levels of these hormones in children's bodies. Although the state and regulatory authorities have been controlling these hormones in food, cosmetics, and veterinary drug residues, these hormones have been continuously ingested by children through various channels and have produced the effect of promoting precocious puberty in the body.

[0007] 2) Plasticizers in the environment, especially phthalates, are ubiquitous in the environment due to their widespread use in making softer and more flexible plastic products. People may be exposed to these compounds through food, air, water, and direct contact with plastic products. The effects of these plasticizers on children's precocious puberty are mainly reflected in several aspects: First, as endocrine disruptors, they can mimic or interfere with hormones in the human body, especially sex hormones such as estrogen and testosterone, which may lead to an imbalance in hormone levels and affect the normal process of sexual maturation. Secondly, plasticizers may have adverse effects on the development of children's reproductive system, such as by prematurely activating the pituitary-gonadal axis, which is an important physiological system that controls sex hormone secretion and sexual maturation. In addition, they may also interfere with the metabolic process of hormones, change the synthesis, secretion, transport, action and excretion of hormones, and thus affect the process of sexual maturation. Finally, because children are more sensitive to environmental pollutants during their growth and development, long-term exposure to an environment containing plasticizers, even at low doses, may lead to a cumulative effect of health problems and increase the risk of precocious puberty.

[0008] 3) There are also bisphenol A compounds. Bisphenol A (BPA) is a large class of compounds and a chemical widely used in the manufacture of plastics and resins, especially in the production of food and beverage containers. The potential health effects of BPA, especially its effects on precocious puberty in children, are mainly related to its characteristics as an endocrine disruptor. BPA can mimic hormones in the human body, especially estrogen, and interfere with normal hormone signaling, which may lead to an imbalance in hormone levels and affect the sexual maturation process of children. In addition, the estrogen-like effects of BPA may have an adverse effect on the development of the reproductive system in children, leading to early changes in sex hormone levels, thereby promoting the occurrence of precocious puberty. BPA may also affect brain development and behavior, especially because the brain is particularly sensitive to hormones, and the disruptive effects of BPA may affect the function of brain areas related to sexual maturation. Considering that children are more sensitive to chemicals in the environment during their growth and development, long-term exposure to an environment containing BPA, even at low doses, may lead to cumulative effects of health problems, including an increased risk of precocious puberty. Although the exact link between BPA and precocious puberty is still under study, there is growing evidence that reducing BPA exposure is essential to protecting children's health and development, so many countries and regions have begun to restrict or ban the use of BPA in children's products. It is precisely because the causes of precocious puberty are very complex and there are many reasons, so if you want to achieve efficient and effective screening for precocious puberty, you must simultaneously detect all of these biomarkers related to precocious puberty.

[0009] 4) At the same time, it is also necessary to detect endogenous hormone compounds related to precocious puberty, and there are more than 20 types of such hormone compounds involved. If all biomarkers that can cause precocious puberty are to be covered, and at the same time, the changes in endogenous hormone compounds that cause precocious puberty in the body are to be covered, the screening of precocious puberty requires up to 102 compounds to be covered.

[0010] 102 early screening biomarkers for sexual precocity were detected at the same time. The compound list is shown in Table 1:

[0011] Table 1

[0012]

[0013]

[0014] Simultaneously measuring multiple biomarkers of precocious puberty in vivo and in vitro presents the following challenges:

[0015] 1) It is difficult to detect 102 compounds simultaneously. It is very difficult to achieve the simultaneous detection of 102 compounds. Furthermore, the simultaneous detection of 102 biomarkers for precocious puberty screening faces the difficulty that these compounds have different structures and properties. When performing separation, analysis and sample pretreatment, it is necessary to take into account the characteristics of the properties of various compounds.

[0016] These 102 compounds have very different properties and each has its own structural characteristics. The following are the structural formulas of several typical compounds.

[0017] a. Structural formula of some hormones derived from food, cosmetics, feed additives, medicines, etc.:

[0018]

[0019] Betamethasone

[0020]

[0021] Diethylstilbestrolv

[0022]

[0023] Stanozolol

[0024] b. Structural formula of some plasticizer metabolites:

[0025]

[0026] Methyl Phthalate

[0027]

[0028] Monopentyl phthalate

[0029]

[0030] c. Structural formula of some bisphenol A compounds:

[0031]

[0032] Bisphenol A

[0033]

[0034] 2,4-Dihydroxybenzophenone

[0035]

[0036] d. Some hormone structures derived from children themselves:

[0037]

[0038] Melatonin

[0039]

[0040] Androsstenedione

[0041]

[0042] Estriol.

[0043] 2) Secondly, the contents of these compounds vary greatly. The detection line of endogenous hormone compounds usually needs to reach picograms per milliliter, while the detection of phthalate metabolites is usually at the nanogram level. In addition, many other compounds that can cause precocious puberty also have different detection lines, so it is necessary to take into account the detection of very low concentration compounds as well as high concentration compounds;

[0044] 3) In addition, although the state and regulatory authorities are testing these compounds that are likely to cause precocious puberty and harm children, they usually test the biomarkers of precocious puberty in food, cosmetics, etc., but these compounds will not exist in their original form after entering the body, but often exist in the form of metabolites. Therefore, only checking the prototypes of these compounds is far from representing the exposure of these compounds in the body. Furthermore, only by testing these biomarkers of precocious puberty in the body can we truly reflect the extent to which a child is exposed to these risks of precocious puberty. Only by accurately testing these biomarkers of precocious puberty in children in vivo can we quickly screen and find the causes that cause or may cause precocious puberty in children and intervene in time. This requires that our detection method must first detect biomarkers of precocious puberty and their metabolites in biological samples, such as plasma or urine.

[0045] 4) Further, although plasma is the most common biological matrix, many in vivo studies use plasma as a biological matrix for testing. However, based on our extensive work, we found that because the plasma matrix is ​​very complex, that is, the plasma biological matrix is ​​"dirty", such a large amount of plasma matrix will cover up these biomarkers of precocious puberty. It is very much like looking for grass under a forest. Therefore, although plasma is a more stable biological matrix, using plasma to screen biomarkers of precocious puberty will reduce the sensitivity of screening. It is possible that precocious puberty has occurred, but because of the complex mechanism of plasma, it covers up such trends and changes. We need to choose a more suitable biological matrix to solve this problem and ensure that the results of the biological matrix are highly consistent with plasma.

[0046] In the prior art, patent document CN113567582A records the application of a biomarker in the screening and diagnosis of precocious puberty in children, which uses high performance liquid chromatography-tandem mass spectrometry to detect the test fluid, and only detects endogenous and exogenous steroid hormone compounds; patent document CN113406254A records a method for simultaneously detecting multiple exogenous and endogenous steroid hormones, which uses high performance liquid chromatography-tandem mass spectrometry to simultaneously detect 54 endogenous and exogenous steroid hormone compounds in human plasma and urine samples, and does not simultaneously detect plasticizers and bisphenol A compounds.

[0047] The above two prior patents only detected hormone compounds, and the number of hormones detected was limited, only 54, which is far from covering the current requirements for the detection of various precocious puberty biomarkers and it is easy to miss key influencing factors.

[0048] Currently, there are no reports on the simultaneous detection of all biomarkers of precocious puberty. Summary of the invention

[0049] In view of this, the purpose of the present invention is to provide a method for simultaneously detecting multiple biomarkers of precocious puberty. The present invention detected a total of 102 biomarkers related to precocious puberty, covering four major categories of different compounds, including exogenous hormones from food, cosmetics, feed additives, medicines, etc., in vivo metabolites of plasticizers, bisphenol A compounds, and endogenous hormone compounds. The present invention can be used for early screening and intervention of precocious puberty in children by detecting the content of compounds or their combinations in urine.

[0050] The objective of the present invention is achieved through the following technical solutions:

[0051] The present invention provides a method for simultaneously detecting multiple biomarkers of precocious puberty, comprising the following steps:

[0052] 1) Take a urine sample and add an internal standard solution;

[0053] 2) Enzymatic hydrolysis: adding glucuronidase solution to the sample solution in step 1) for enzymatic hydrolysis;

[0054] 3) After enzymatic hydrolysis, the samples were loaded onto an HLB solid phase extraction plate and a MAX solid phase extraction plate, respectively. After the interfering substances were washed away with an eluent, the samples were eluted with an eluent. The eluents from the HLB solid phase extraction plate and the MAX solid phase extraction plate were combined and tested by UPLC-MS to obtain the contents of various biomarkers of precocious puberty.

[0055] The multiple biomarkers of precocious puberty include exogenous hormone compounds, plasticizers, bisphenol A compounds, and endogenous hormone compounds;

[0056] The number of detection types of the multiple precocious puberty biomarkers is 102 or more.

[0057] In step 2), the concentration of the glucuronidase solution is 5,000-20,000 U / mL.

[0058] In step 2), the enzymatic hydrolysis time is 1-2 hours and the temperature is 35-60°C.

[0059] In step 2), the volume ratio of the glucuronidase solution to the sample solution should be 20:1-40:1.

[0060] In step 3), the eluent is a mixed solution comprising formic acid and an organic phase, wherein the mass fraction of formic acid in the mixed solution is 2-3%; the organic phase comprises acetonitrile and methanol, and the volume ratio of the two is 9:1-8:2.

[0061] In step 3), the eluent is water.

[0062] The internal standard solution contains 20 stable isotope internal standards of endogenous hormone compounds and 11 stable isotope internal standards of plasticizer metabolites;

[0063] The 20 endogenous hormone compounds include: 11-deoxycorticosterone, 17α-hydroxyprogesterone, 21-deoxycortisol, corticosterone, 11-deoxycortisol, androstenedione, dihydrotestosterone, testosterone, pregnenolone, progesterone, melatonin, cortisol, cortisone, dexamethasone, aldosterone, 17α-hydroxypregnenolone, estrone, estradiol, estriol, and dehydroepiandrosterone sulfate;

[0064] The 11 plasticizer metabolites include: monomethyl phthalate, monoethyl phthalate, monophenyl phthalate, mono-n-butyl phthalate, mono(3-hydroxy-n-butyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, mono(3-carboxypropyl) phthalate, monoisobutyl phthalate, and mono(2-hydroxy-isobutyl) phthalate.

[0065] In step 1), the concentration of the internal standard solution is 2-500 ng / mL.

[0066] The internal standard solution is used to calibrate the errors that may be introduced into the entire operation, as well as the detector response and matrix effects.

[0067] Urine is combined with enzymatic treatment to take urine as the biological matrix for early screening of precocious puberty, and the measurement results are as close to blood as possible; in urine, hormones and bisphenol A compounds are reduced to prototype compounds by enzymatic hydrolysis, and plasticizer compounds are first converted into primary metabolites in the body and cannot be restored to the prototype by enzymatic hydrolysis. Only the secondary metabolites of plasticizers can be converted into primary metabolites, and the content of prototypes in blood is reflected by detecting the primary metabolites of plasticizers in urine.

[0068] All early screening biomarkers for precocious puberty were purified and enriched by combining HLB+MAX solid phase extraction columns, in which the HLB solid phase extraction column was responsible for purifying compounds that were easily retained on C18, and the MAX solid phase extraction column was responsible for purifying negatively charged compounds in early screening biomarkers for precocious puberty.

[0069] In step 3), when the UPLC-MS is used for analysis, the liquid phase analysis conditions used are:

[0070] The chromatographic column is ACQUITY UPLC BEH Phenyl. A chromatographic column with phenyl groups bonded to the column was selected to be compatible with all biomarkers for early screening of precocious puberty.

[0071] Mobile phase A: 0.1% ammonium fluoride in formic acid, pH 2.5-4.5;

[0072] Mobile phase B: acetonitrile:methanol volume ratio 9:1;

[0073] The mobile phase gradient conditions were:

[0074]

[0075]

[0076] Mass spectrometry analysis conditions:

[0077] Ion source: ESI ion source

[0078] Detection mode: MRM acquisition mode;

[0079] The ion source parameters are: cone voltage: 30 V; ion source temperature: 500 °C; desolvation temperature: 150 °C;

[0080] MRM monitoring information is:

[0081]

[0082]

[0083]

[0084] As one embodiment of the present invention, the multiple biomarkers of precocious puberty include:

[0085]

[0086]

[0087]

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] 1. In the prior art, blood matrix is ​​usually selected, such as plasma or serum, but a large amount of proteins, lipids, amino acids, etc. in plasma and serum will mask the signals of precocious puberty biomarkers; however, in the prior art, when the traditional detection method HLB (or C18) solid phase extraction method is used, if urine matrix is ​​selected, there is no suitable method to trace the detection results in urine to blood, reflecting the precocious puberty biomarkers in blood; the present invention uses urine as a biological matrix and solves the problem of tracing to blood in two steps: 1) by enzymatic hydrolysis (optimized enzymatic hydrolysis dosage and conditions); the biomarker that forms a specific binding compound is converted back to the prototype; 2) the biomarker that has been metabolized into metabolites in the blood, that is, the biomarker that can no longer be restored by enzymatic hydrolysis, is traced to the blood by detecting the metabolites of the biomarker, reflecting the content of the biomarker in the blood;

[0090] 2. The traditional method uses HLB (or C18) solid phase extraction to process hormone compounds, and uses MAX (or anion and cation) solid phase extraction to process plasticizer compounds, but these two methods cannot take into account each other; they can only separate a part of the compounds; the present invention uses parallel SPE method to perform the operations of these two methods in parallel during sample processing, and mixes the samples in the last step, so as to achieve the fusion and concentration of all target compounds to be detected before injection; and take into account the analysis requirements of the four major types of compounds;

[0091] 3. The mobile phase of the traditional method usually achieves better separation or sensitivity by adding formic acid, ammonium formate, ammonium acetate and other acids or salts, but these conventional methods cannot meet the needs of detecting these precocious puberty target compounds; the present invention 1) through the investigation and optimization of various chromatographic columns, can only use phenyl columns to be compatible with 4 major types of compounds; 2) the mobile phase B (organic phase) uses methanol: acetonitrile mixed mobile phase, and the mobile phase A (aqueous phase) adds ammonium fluoride, and the pH value is adjusted to 2.5-4.5; the separation of the compounds is achieved; thereby taking into account the separation and sensitivity requirements of all compounds;

[0092] 4. The present invention realizes the simultaneous detection of at least 102 steroid hormones, plasticizers, and bisphenol A compounds in the same sample by selecting chromatographic columns and mobile phases suitable for the above 102 compounds, covering sources such as medicines, foods, and the environment. The present invention is easy to operate and only takes 11 minutes to analyze, which greatly improves the detection speed, throughput, and types and numbers of precocious puberty biomarkers covered, and can quickly and comprehensively evaluate various organisms, patients, and healthy people. In the future, it can be used to accurately determine the content of steroid hormones, plasticizers, and bisphenol A compounds in urine.

[0093] 5. The present invention measured urine samples of 30 children, and the results showed that this method can effectively screen children for biomarkers of precocious puberty;

[0094] 6. The present invention uses the analysis results in urine samples to reflect the analysis results of precocious puberty in children, thereby preventing the large matrix effect of plasma samples from interfering with the analysis results, reducing the detection workload, and improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0096] Figure 1 is a chromatogram of 102 biomarkers in the present invention;

[0097] Figure 2is a Bland-Altman plot of urine biomarkers and blood biomarkers in Example 3;

[0098] Figure 3 Standard curve for hormones (prednisone) from food, cosmetics, feed additives, and medicines;

[0099] Figure 4 This is the standard curve of plasticizer (2,4-dihydroxybenzophenone);

[0100] Figure 5 The standard curve of bisphenol A (2,4-dihydroxybenzophenone);

[0101] Figure 6 The standard curve is derived from the child's own hormone (aldosterone);

[0102] Figure 7 It is a flow chart of the technical solution in the present invention;

[0103] Figure 8 This is a schematic diagram of the present invention using urine as a biological matrix and a two-step method to solve the problem of tracing the source to blood. DETAILED DESCRIPTION

[0104] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0105] In the following embodiments:

[0106] HLB solid phase extraction plate: Yujing Technology Purphy HLB

[0107] MAX solid phase extraction plate: Purphy MAX from Yujing Technology;

[0108] The stable isotope internal standard solution contains 20 stable isotope internal standards of endogenous hormones and 11 stable isotope internal standards of plasticizer metabolites; the concentration of the internal standard solution is 500 ng / mL.

[0109] Among them, 20 endogenous hormone compounds include 11-deoxycorticosterone, 17α-hydroxyprogesterone, 21-deoxycortisol, corticosterone, 11-deoxycortisol, androstenedione, dihydrotestosterone, testosterone, pregnenolone, progesterone, melatonin, cortisol, cortisone, dexamethasone, aldosterone, 17α-hydroxypregnenolone, estrone, estradiol, estriol, and dehydroepiandrosterone sulfate; the concentration of each endogenous hormone is 20ng / mL. 11 plasticizer metabolites include o- Monomethyl phthalate, monoethyl phthalate, monophenyl phthalate, mono-n-butyl phthalate, mono(3-hydroxy-n-butyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, mono(3-carboxypropyl) phthalate, monoisobutyl phthalate, mono(2-hydroxy-isobutyl) phthalate;

[0110] Among them, the concentration of each plasticizer metabolite was 200ng / mL.

[0111] The concentration units of hormones mentioned throughout the text are all ng / mL.

[0112] Example 1 Establishment of a standard curve for the detection of 102 compounds in urine

[0113] The purpose of this example is to investigate the lower limit of quantitation, upper limit of quantitation, standard curve and regression equation of 102 compounds.

[0114] The specific steps are as follows:

[0115] 1. Take 200 μl of standard curve working solution;

[0116] Preparation of standard curve working solution: Prepare standard working solutions of different concentrations of 102 compounds in sequence: 0.008 ng / mL, 0.021 ng / mL, 0.043 ng / mL, 0.086 ng / mL, 0.217 ng / mL,

[0117] 0.434ng / mL, 0.869ng / mL, 2.174ng / mL, 4.434ng / mL, 8.696ng / mL, 21.74ng / mL, 43.480ng / mL, 86.96ng / mL, 217.4ng / mL, 434.8ng / mL, 869.6ng / mL.

[0118] 2. Add 100 μl of stable isotope internal standard solution to each standard curve working solution. The internal standard solution is used to calibrate the error that may be introduced by the entire operation, as well as the response and matrix effect of the detector;

[0119] 3. Add glucuronidase and perform enzymolysis at 35°C for 1 hour; wherein:

[0120] The concentration of the glucuronidase solution was 5,000 U / mL;

[0121] The volume ratio of glucuronidase solution to standard curve working solution was 40:1.

[0122] 4. Load the enzymatically hydrolyzed samples onto HLB solid phase extraction plates and MAX solid phase extraction plates, respectively, with 250 μL on each plate; add water for rinsing and then elute with eluent, the eluent is a mixed solution containing formic acid and an organic phase, the mass fraction of formic acid in the mixed solution is 2%; the organic phase includes acetonitrile and methanol, and the volume ratio of the two is 8:2.

[0123] 5. Combine and collect the eluates from the HLB solid phase extraction plate and the MAX solid phase extraction plate in step 4, take 50 μL of the eluate and add 100 μL of water to dilute; vortex mix and inject into the UPLC / MS mass spectrometer for detection and analysis;

[0124] UPLC-MS analysis of samples

[0125] The liquid phase analysis conditions used were:

[0126] Column: ACQUITY UPLC BEH Phenyl

[0127] Mobile phase A: 0.1% ammonium fluoride in formic acid, pH 4.5;

[0128] Mobile phase B: acetonitrile:methanol volume ratio 9:1;

[0129] The mobile phase gradient conditions are shown in Table 2:

[0130] Table 2

[0131]

[0132]

[0133] Mass spectrometry analysis conditions:

[0134] Ion source: ESI ion source

[0135] Detection mode: MRM acquisition mode;

[0136] The ion source parameters are: cone voltage: 30 V; ion source temperature: 500 °C; desolvation temperature: 150 °C;

[0137] MRM monitoring information is shown in Table 3:

[0138] Table 3

[0139]

[0140]

[0141]

[0142] The lower and upper limits of quantitation of the 102 compounds are shown in Table 4:

[0143] Table 4

[0144]

[0145]

[0146]

[0147] The standard curves and standard curve regression equations of 102 compounds are shown in Table 5:

[0148] Table 5

[0149]

[0150]

[0151]

[0152]

[0153] Figure 3 Standard curve for hormones (prednisone) from food, cosmetics, feed additives, and medicines;

[0154] Figure 4 This is the standard curve of plasticizer (2,4-dihydroxybenzophenone);

[0155] Figure 5 The standard curve of bisphenol A (2,4-dihydroxybenzophenone);

[0156] Figure 6 This is a standard curve of hormone (aldosterone) derived from children themselves.

[0157] Example 2 Method for Simultaneously Determining Multiple Exogenous and Endogenous Precocious Puberty Hormones in Urine

[0158] Sample: Urine collected from 30 children at the same time.

[0159] Test method:

[0160] Step 1: Take 200 μL of urine sample from each specimen;

[0161] Step 2: Add 100 μL of stable isotope internal standard solution to each urine sample. The internal standard solution is used to calibrate the error that may be introduced by the entire operation, as well as the response of the detector and the matrix effect.

[0162] Step 3: Add glucuronidase solution to convert the metabolites of hormones, bisphenol A and plasticizers in urine into the prototype of hormones, the prototype of bisphenol A and the primary metabolites of plasticizers; perform enzymatic hydrolysis at 35°C for 1 hour to cut the chemical bonds of the bound metabolites;

[0163] in:

[0164] The concentration of the glucuronidase solution was 5,000 U / mL;

[0165] The volume ratio of the glucuronidase solution to the sample solution was 40:1.

[0166] Step 4: Load the enzymatically hydrolyzed urine onto an HLB solid phase extraction plate and a MAX solid phase extraction plate, respectively, with 250 μL loaded onto each plate; add water for rinsing and then elute with an eluent, the eluent being a mixed solution comprising formic acid and an organic phase, wherein the mass fraction of formic acid in the mixed solution is 2%; the organic phase comprises acetonitrile and methanol, and the volume ratio of the two is 8:2.

[0167] Step 5: Combine and collect the eluates from the HLB solid phase extraction plate and the MAX solid phase extraction plate in step 4, take 50 μL of the eluate and add 100 μL of water to dilute; vortex mix and inject into the UPLC / MS mass spectrometer for detection and analysis.

[0168] The liquid phase analysis conditions used were:

[0169] Column: ACQUITY UPLC BEH Phenyl

[0170] Mobile phase A: 0.1% ammonium fluoride in formic acid, pH 4.5;

[0171] Mobile phase B: acetonitrile:methanol volume ratio 9:1;

[0172] The mobile phase gradient conditions are the same as those in Table 2 in Example 1:

[0173] Mass spectrometry analysis conditions:

[0174] Ion source: ESI ion source

[0175] Detection mode: MRM acquisition mode;

[0176] The ion source parameters are: cone voltage: 30 V; ion source temperature: 500 °C; desolvation temperature: 150 °C;

[0177] The MRM monitoring information is the same as Table 3 in Example 1 (the detected results are compared with the calibration curve equations of Example 1 to obtain the data in Tables 6-8):

[0178] The urine test results of 30 children are shown in Tables 6-8:

[0179] Table 6

[0180]

[0181]

[0182]

[0183]

[0184] Table 7

[0185]

[0186]

[0187]

[0188]

[0189] Table 8

[0190]

[0191]

[0192]

[0193] Note: ND in the table means not detected.

[0194] Example 3: Consistency test of early screening biomarkers for precocious puberty in urine and corresponding compounds in blood

[0195] The purpose of this embodiment is to examine the invention's use of urine to present early screening biomarkers for precocious puberty in the blood and their consistency with blood. Because the matrix in the blood is relatively complex and masks many biomarkers, a portion of typical biomarkers that are not masked are compared to complete the investigation.

[0196] Among them, the biomarkers masked in plasma include at least prednisone, methyltestosterone, androstenedione, progesterone, and testosterone.

[0197] Samples: Blood and urine samples from 40 children at the same time.

[0198] Step 1: Take 200 μl of urine and plasma samples (or standard curve, quality control) from each sample;

[0199] Step 2: Add 100 μl of stable isotope internal standard solution to each urine and plasma sample (or standard curve, quality control). The internal standard solution is used to calibrate the error that may be introduced by the entire operation, as well as the response of the detector and the matrix effect.

[0200] Step 3: Add glucuronidase solution to convert the metabolites of hormones, bisphenol A and plasticizers in urine or plasma into the prototype of hormones, the prototype of bisphenol A and the primary metabolites of plasticizers; perform enzymatic hydrolysis at 35°C for 1 hour to cut the chemical bonds of the bound metabolites;

[0201] in:

[0202] The concentration of the glucuronidase solution was 5,000 U / mL;

[0203] The volume ratio of the glucuronidase solution to the sample solution was 40:1.

[0204] Step 4: Load the enzymatically hydrolyzed urine onto an HLB solid phase extraction plate and a MAX solid phase extraction plate, respectively, with 250 μL loaded onto each plate; add water for rinsing and then elute with an eluent, the eluent being a mixed solution comprising formic acid and an organic phase, wherein the mass fraction of formic acid in the mixed solution is 2%; the organic phase comprises acetonitrile and methanol, and the volume ratio of the two is 8:2.

[0205] Step 5: Combine and collect the eluates from the HLB solid phase extraction plate and the MAX solid phase extraction plate in step 4, take 50 μL of the eluate and add 100 μL of water to dilute; vortex mix and inject into the UPLC / MS mass spectrometer for detection and analysis.

[0206] The liquid phase analysis conditions used were:

[0207] Column: ACQUITY UPLC BEH Phenyl

[0208] Mobile phase A: 0.1% ammonium fluoride in formic acid, pH 4.5;

[0209] Mobile phase B: acetonitrile:methanol volume ratio 9:1;

[0210] The mobile phase gradient conditions are the same as those in Table 2 in Example 1:

[0211] Mass spectrometry analysis conditions:

[0212] Ion source: ESI ion source

[0213] Detection mode: MRM acquisition mode;

[0214] The ion source parameters are: cone voltage: 30 V; ion source temperature: 500 °C; desolvation temperature: 150 °C;

[0215] MRM monitoring information is the same as Table 9-16 in the embodiment:

[0216] Table 9

[0217]

[0218]

[0219] Table 10

[0220]

[0221]

[0222] Table 11

[0223]

[0224]

[0225] Table 12

[0226]

[0227] Table 13

[0228]

[0229]

[0230] Table 14

[0231]

[0232]

[0233] Table 15

[0234]

[0235]

[0236] Table 16

[0237]

[0238] The results of plasma determination are shown in Tables 17-24 (selected representative compounds):

[0239] Table 17

[0240]

[0241] Table 18

[0242]

[0243]

[0244] Table 19

[0245]

[0246] Table 20

[0247]

[0248]

[0249] Table 21

[0250]

[0251] Table 22

[0252]

[0253] Table 23

[0254]

[0255]

[0256] Table 24

[0257]

[0258]

[0259] The test results of plasma and urine involve multiple indicators, so Bland-Altman analysis is selected to analyze the consistency of plasma and urine test results. Bland-Altman analysis, also known as difference-mean analysis, is a statistical method used to evaluate the consistency of two different measurement methods. Figure 2 As shown, the results of urine biomarkers measured by this method are consistent with those in plasma, and can well demonstrate the characteristics of early screening biomarkers for precocious puberty in the blood.

[0260] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for simultaneously detecting multiple biomarkers of precocious puberty, characterized in that: The steps include: 1) Take a urine sample and add an internal standard solution; 2) Enzymatic hydrolysis: adding glucuronidase solution to the sample solution of step 1) for enzymatic hydrolysis; performing enzymatic hydrolysis at 35° C. for 1 hour; wherein: the concentration of the glucuronidase solution is 5,000 U / mL; and the volume ratio of the glucuronidase solution to the standard curve working solution is 40:1; 3) The samples after enzymatic hydrolysis in step 2) are loaded onto an HLB solid phase extraction plate and a MAX solid phase extraction plate, respectively, with 250 μL loaded onto each plate; after rinsing with water, the samples are eluted with an eluent, wherein the eluent is a mixed solution comprising formic acid and an organic phase, wherein the mass fraction of formic acid in the mixed solution is 2%; the organic phase comprises acetonitrile and methanol, and the volume ratio of the two is 8:2; the eluents of the HLB solid phase extraction plate and the MAX solid phase extraction plate are collected and combined, and 50 μL of the eluent is diluted with 100 μL of water; after vortex mixing, the eluent is injected into a UPLC / MS mass spectrometer for detection and analysis to obtain the contents of multiple biomarkers of precocious puberty; The multiple biomarkers of precocious puberty include exogenous hormone compounds, plasticizers, bisphenol A compounds, and endogenous hormone compounds; The multiple biomarkers of precocious puberty include: The number of detection types of the multiple precocious puberty biomarkers is 102 or more; In step 3), when the UPLC-MS is used for analysis, the liquid phase analysis conditions used are: chromatographic column: ACQUITYUPLC BEH Phenyl Mobile phase A: 0.1% ammonium fluoride in formic acid, pH 2.5-4.5; Mobile phase B: acetonitrile:methanol volume ratio 9:1; The mobile phase gradient conditions were: Mass spectrometry analysis conditions: Ion source: ESI ion source Detection mode: MRM acquisition mode; The ion source parameters are: cone voltage: 30v; ion source temperature: 500℃; desolvation temperature: 150℃; MRM monitoring information is:

2. The method according to claim 1, characterized in that: In step 1), the internal standard solution contains 20 stable isotope internal standards of endogenous hormone compounds and 11 stable isotope internal standards of plasticizer metabolites; The 20 endogenous hormone compounds include: 11-deoxycorticosterone, 17α-hydroxyprogesterone, 21-deoxycortisol, corticosterone, 11-deoxycortisol, androstenedione, dihydrotestosterone, testosterone, pregnenolone, progesterone, melatonin, cortisol, cortisone, dexamethasone, aldosterone, 17α-hydroxypregnenolone, estrone, estradiol, estriol, and dehydroepiandrosterone sulfate; The 11 plasticizer metabolites include: monomethyl phthalate, monoethyl phthalate, monophenyl phthalate, mono-n-butyl phthalate, mono(3-hydroxy-n-butyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, mono(3-carboxypropyl) phthalate, monoisobutyl phthalate, and mono(2-hydroxy-isobutyl) phthalate.

3. The method according to claim 1, characterized in that In step 1), the concentration of the internal standard solution is 2-500 ng / mL.

Images