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CN116448901A - Endocrine disrupter for screening sexual precocity risk of children and application thereof - Google Patents

Endocrine disrupter for screening sexual precocity risk of children and application thereof Download PDF

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Publication number
CN116448901A
CN116448901A CN202310191749.8A CN202310191749A CN116448901A CN 116448901 A CN116448901 A CN 116448901A CN 202310191749 A CN202310191749 A CN 202310191749A CN 116448901 A CN116448901 A CN 116448901A
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screening
solution
kit
children
sexual precocity
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Inventor
薛斌
覃健
殷梓晨
李妍
袁玉山
沈宁
徐滔
马培旗
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Sir Run Run Hospital Nanjing Medical University
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Sir Run Run Hospital Nanjing Medical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention provides an endocrine disrupter for screening children sexual precocity risk and application thereof, belonging to the technical field of biochemical detection; in the invention, one or a combination of six endocrine disruptors of cyproconazole, dimethoate, chloropyrimidol, benzopyrene, cadmium and mercury is used as a marker for screening the risk of children sexual precocity, the endocrine disruptors are detected by adopting a high performance liquid chromatography tandem mass spectrometry detection technology, and the endocrine disruptors are prepared into a kit for analyzing the risk of children sexual precocity; beta-glucosidase/arylsulfatase and catechin are added into the kit to improve the sensitivity and accuracy of the kit, and the detection limit of the kit is reduced by regulating and controlling the mass spectrum detection conditions, so that the risk of sexual precocity of children can be screened early.

Description

Endocrine disrupter for screening sexual precocity risk of children and application thereof
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to an endocrine disrupter for screening children sexual precocity risk and application thereof.
Background
The sexual precocity of children is a adolescent dysplasia disease, namely that male boys are 9 years old and girls are 8 years old, second sexual feature development appears, the epiphyseal closure is shown, the menstruation is early, the height of the adult is lower than that of normal people, meanwhile, the intelligence and sexual psychology are immature due to the premature development of the sexual feature of the children, psychological and physiological disorder is caused, and serious negative effects are brought to families and society. Thus, there is a need for early diagnosis or prevention of premature sexual functioning in children to reduce their clinical incidence.
At present, clinical diagnosis is mainly based on the advanced appearance of secondary sex characteristics of patients, imaging examination and combination diagnosis of various indexes such as serum neutral hormone (such as follicle stimulating hormone, luteinizing hormone, prolactin, estradiol, testosterone and progesterone) molecular diagnosis markers, but the indexes are all started to appear after the occurrence of sexual precocity of children and cannot be used as screening markers of early onset risk factors. In the prior art, the wrist bone age index can be used for diagnosing the children sexual precocity, but the bone age index can be used for effectively diagnosing the idiopathic central precocity, but the significance of diagnosing the peripheral precocity is not great, so that the method can only cover a smaller part of patient population and is difficult to be used for screening the morbidity risk factors. The female estrogen receptor gene locus diversity is also related to sexual precocity, the mutant genes increase the risk of sexual precocity of the female, but the estrogen receptor gene polymorphism is not a specific cause of sexual precocity of the female, and can be influenced by other disease factors, so that the screening result is not completely accurate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an endocrine disrupter for screening the risk of sexual precocity of children and application thereof. In the invention, one or a combination of six endocrine disruptors of cyproconazole, dimethoate, chloropyrimidol, benzopyrene, cadmium and mercury is used as a marker for screening the risk of children sexual precocity, the endocrine disruptors are detected by adopting a high performance liquid chromatography tandem mass spectrometry detection technology, and the endocrine disruptors are prepared into a kit for analyzing the risk of children sexual precocity; beta-glucosidase/arylsulfatase and catechin are added into the kit to improve the sensitivity and accuracy of the kit, and the detection limit of the kit is reduced by regulating and controlling the mass spectrum detection conditions, so that the risk of sexual precocity of children can be screened early.
The invention provides an endocrine disrupter for screening children sexual precocity risk, comprising: one or more combinations of cyproconazole, dimethoate, chloropyrinol, benzopyrene, cadmium and mercury.
Wherein the premature sexual desire of the child is caused by chemical toxins in the environment.
The invention also provides application of the endocrine disruptors in preparing a reagent or a kit for screening early childhood sexual precocity caused by chemical toxins in the environment.
The invention also provides a kit for screening early childhood sexual precocity, comprising: the urine sample pretreatment reagent combination, eluent, mixed standard working solution, diluent, eluent/activating solution and compound solution; the urine sample pretreatment reagent combination comprises acetic acid-sodium acetate buffer solution, beta-glucuronidase/arylsulfatase and catechin.
Wherein, the dosage ratio of the acetic acid-sodium acetate buffer solution, beta-glucuronidase/arylsulfatase to catechin is 2mL to 10 mu L to 5mg; the acetic acid-sodium acetate buffer solution is formed by mixing 0.2M acetic acid aqueous solution and 0.2M sodium acetate aqueous solution according to the volume ratio of 1:6.
The mixed standard working solution is acetonitrile solution containing endocrine disruptors, wherein the endocrine disruptors are cyproconazole, dimethoate, chloropyrimidol, cadmium and mercury with the concentration of 1 mug/mL and benzopyrene with the concentration of 1ng/mL;
the diluent is an aqueous solution containing 2g/mL urea and 1g/mL sodium chloride;
the leaching liquid/activating liquid is water;
the eluent/activating solution is acetonitrile;
the complex solution is acetonitrile water solution containing 0.1% (v/v) formic acid, and the volume ratio of acetonitrile to water is 1:9.
The invention also provides a use method of the kit, which comprises the following steps:
(1) Collecting urine samples of normal children and precocious puberty children, and treating the urine samples by using a urine sample pretreatment reagent combination;
(2) Preparing a mixed standard substance solution with a concentration gradient by using a mixed standard substance working solution in the kit;
(3) Performing LC-MS/MS mass spectrum sample injection on-machine detection on the prepared sample to be detected and the standard solution;
(4) And drawing a standard curve of the mixed standard substance solution according to the detection result, and screening the risk of the children sexual precocity morbidity.
Wherein, the cyproconazole, dimethoate, chlorpyrimidol and benzopyrene are detected by adopting a liquid chromatography tandem mass spectrometer, and the mass spectrum conditions are as follows:
the MRM parameters are:
the cadmium and mercury are analyzed by an inductively coupled plasma mass spectrometer (ICP-MS), and mass spectrum conditions are as follows:
high frequency power: 1.2kW
Auxiliary gas flow rate: 1.1L/min
Sampling depth: 5.0mm
Plasma gas flow rate: 8.0L/min
Carrier gas flow rate: 0.7L/min
Mist room temperature: 5 DEG C
High frequency: 27.12MHz
Collision gas type: he (He)
Cell voltage: -21V
Collision gas flow rate: 6.0mL/min
Energy filter voltage: 5.0V.
Compared with the prior art, the invention has the beneficial effects that:
based on the current situation that chemical toxins in the environment are relatively high in the pathogenesis of the premature sexual life of children, one or more of cyproconazole, dimethoate, chlorpyrimidol, bisphenol A, cadmium and mercury endocrine disruptors are used as markers for screening the risk of the premature sexual life of children caused by the chemical toxins in the environment, and the markers are prepared into a kit for screening the premature sexual life of early children. From the etiology perspective, the marker and the diagnostic kit for monitoring the disease factors causing the disease before the disease is developed have important clinical value for early screening of the risk of sexual precocity of children.
The kit is prepared based on one or more of cyproconazole, dimethoate, chloropyrimidol, bisphenol A, cadmium and mercury endocrine disruptors, has the advantages of simple sample acquisition, low detection limit, good repeatability, high screening sensitivity and specificity and the like, and overcomes the limitations of one-sided property and hysteresis, complex detection method, high sample acquisition difficulty and the like of the traditional clinical screening of the children precocity.
According to the invention, the sensitivity and the accuracy of the kit are improved by adding beta-glucuronidase/arylsulfatase and catechin into the urine sample pretreatment reagent combination, and the detection limit of the kit is reduced by regulating and controlling the mass spectrum detection condition, so that the risk of premature sexual intercourse of children can be screened early.
Drawings
FIG. 1 is a standard graph of cyproconazole.
Fig. 2 is a standard graph of dimethoate.
FIG. 3 is a standard graph of chloropyrimidol.
FIG. 4 is a standard graph of benzopyrene.
FIG. 5 is a standard graph of cadmium.
Fig. 6 is a standard graph of mercury.
FIG. 7 shows the results of testing of urine samples from cyproconazole (a), dimethoate (b), chloropyrimidol (c), benzopyrene (d), cadmium (e) and mercury (f) in the normal control group and in the pediatric precocity group.
FIG. 8 is a Spearman rank correlation analysis of onset of precocity with cyproconazole, dimethoate, clopyrimidinol, benzopyrene, cadmium and mercury.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
Example 1: preparation of kit for screening children for sexual precocity
The components of the kit for screening children for precocity and the preparation of the kit are as follows:
urine sample pretreatment reagent combination: acetic acid-sodium acetate buffer, beta-glucuronidase/arylsulfatase, catechin; wherein the acetic acid-sodium acetate buffer solution is prepared by mixing 0.2M acetic acid aqueous solution and 0.2M sodium acetate aqueous solution according to the volume ratio of 1:6.
Mixing standard working solution: acetonitrile is used as a solvent, and the concentrations of the solute cyproconazole, dimethoate, chloropyrimidol, cadmium and mercury are all 1 mug/mL, and the concentration of benzopyrene is 1ng/mL. The preparation method comprises the following steps: 10mg of cyproconazole, dimethoate, chloropyrimidol, cadmium and mercury respectively and 10ng of benzopyrene are fixed to 5mL by acetonitrile to obtain 6 standard substance solutions respectively, 10 mu L of each standard substance solution is mixed, and then acetonitrile is used for diluting the mixed standard substance working solution to corresponding concentration.
Eluent: phase A: 0.1% formic acid in water, phase B: acetonitrile solution of 0.1% formic acid
Dilution liquid: urine matrix diluent, specifically an aqueous solution containing 2g/mL urea and 1g/mL sodium chloride;
eluent/activator: water;
eluent/activator: acetonitrile;
and (3) a complex solution: an aqueous acetonitrile solution containing 0.1% (v/v) formic acid, the volume ratio of acetonitrile to water was 1:9.
Example 2: method for screening kit for premature sexual intercourse of children
(1) Treatment of the solution to be tested:
2mL of urine sample is filtered by a 0.22 mu m filter membrane, acetic acid-sodium acetate buffer solution is added to adjust the pH value to 5.4, then 10 mu L of beta-glucuronidase/arylsulfatase is added to specifically degrade substances to be detected which are glucuronidated or sulfolated in the urine sample, then 5mg of catechin is added, and the urine sample is incubated overnight at room temperature. The enzymatically hydrolyzed sample was subjected to solid phase extraction with SPE cartridge (C18 ENVI,0.25 g), and the extract was eluted with 2mL of acetonitrile, blow-dried with nitrogen, and redissolved with 100. Mu.L of 0.1% formic acid in acetonitrile water to give the solution to be tested.
(2) Drawing a standard curve:
respectively transferring a proper amount of standard solution from the mixed standard working solution, and diluting the standard solution into 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 1ng/mL and 0.5ng/mL (cyproconazole, dimethoate, chlorpyrimidol, cadmium and mercury) by using a urine matrix diluent according to different detection indexes; 100pg/mL, 50pg/mL, 20pg/mL, 10pg/mL, 5pg/mL, 1pg/mL, 0.5pg/mL (benzopyrene) and then tested under chromatographic and mass spectrometry conditions as described below, and after the standard curve is drawn, the actual sample can be quantified using the standard curve.
Analysis of cyproconazole, dimethoate, chlorophenylpyrimidinol and benzopyrene by liquid chromatography tandem mass spectrometer (LC-MS/MS)
The chromatographic conditions are as follows:
mobile phase: phase A: 0.1% formic acid in water, phase B: 0.1% formic acid in acetonitrile;
chromatographic column: ACQUITY UPLC BEH C18 chromatographic column;
elution mode: gradient elution and gradient elution procedure table are shown in table 1.
TABLE 1 gradient elution program table
Time(min) Phase A (%) Phase B (%)
1 95 5
6 5 95
7 5 95
7.1 65 35
10 Stop
The mass spectrum conditions are as follows:
scanning mode: MRM (MRM parameters are shown in Table 2).
Table 2.Mrm parameters
2 analysis of cadmium and mercury by inductively coupled plasma Mass Spectrometry (ICP-MS)
The mass spectrum conditions are as follows:
high frequency power: 1.2kW
Auxiliary gas flow rate: 1.1L/min
Sampling depth: 5.0mm
Plasma gas flow rate: 8.0L/min
Carrier gas flow rate: 0.7L/min
Mist room temperature: 5 DEG C
High frequency: 27.12MHz
Collision gas type: he (He)
Cell voltage: -21V
Collision gas flow rate: 6.0mL/min
Energy filter voltage: 5.0V
In table 2, the precursor ions are characteristic ions generated by endocrine disruptors in a primary Mass Spectrometry (MS) mode, also called parent ions; the product ions are characteristic fragment ions, also called daughter ions, generated by the parent ions generated by endocrine disruptors after being collided by inert gas (such as argon) in a secondary (MS/MS) mass spectrum mode.
According to the detection results, a standard curve is drawn, and fig. 1 is a standard curve of cyproconazole, and as can be seen from the figure, the equation of the standard curve is y=12103x+2588.1, r= 0.9992, and the detection limit is 0.1ng/mL.
Fig. 2 is a standard plot of dimethoate, and as can be seen from the graph, the equation of the standard plot is y=58108x+4238.3, r=0.9999, and the detection limit is 0.1ng/mL.
Fig. 3 is a standard graph of chloropyrimidol, and as can be seen from the graph, the equation of the standard graph is y=248X-49.4, r=0.9997, and the detection limit is 0.1ng/mL.
Fig. 4 is a standard graph of benzopyrene, and as can be seen from the graph, the equation of the standard graph is y=236.11x+11.74, r=0.9990, and the detection limit is 0.1pg/mL.
Fig. 5 is a standard graph of cadmium, from which it can be seen that the equation of the standard graph is y= 1.9852X-0.2491, r=0.9999, and the detection limit is 0.1ng/mL.
Fig. 6 is a standard graph of mercury, from which it can be seen that the equation for the standard graph is y= 0.6574X-0.1823, r= 0.9996, and a detection limit of 0.1ng/mL.
In the embodiment, a trimethyl silanization derivative combined GC-MS method is used as a control group, and the detection method in the invention is used as an experimental group to detect the cyproconazole, dimethoate, chloropyrimidol and benzopyrene with the theoretical concentration of 100ng/mL respectively; the detection recovery rates of cyproconazole, dimethoate, chlorpyrinol, benzopyrene, cadmium and mercury are respectively compared by taking an inductively coupled plasma emission spectrometry (ICP-OES) method as a control group and taking a detection method in the invention as an experimental group, wherein the theoretical concentration of the cadmium and the mercury is 100ng/mL, and the comparison results are shown in Table 3.
TABLE 3 recovery of cyproconazole, dimethoate, chloropyrimidinol, benzopyrene, cadmium and Mercury by different detection methods
Control group Experimental group
Concentration of cyproconazole (ng/mL) 84.85 103.93
Recovery of cyproconazole (%) 85 104
Concentration of Dimethoate (ng/mL) 91.28 104.48
Recovery of Dimethoate (%) 91 105
Concentration of Chloropyrimidinol (ng/mL) 117.92 107.90
Recovery of Chloropyrimidinol (%) 118 108
Concentration of benzopyrene (pg/mL) 82.32 91.38
Recovery of benzopyrene (%) 82 91
Cadmium concentration (ng/mL) 114.49 106.93
Recovery of cadmium (%) 115 107
Concentration of mercury (ng/mL) 86.99 91.49
Recovery of mercury (%) 87 92
The recovery rate is the ratio of the average value of the measured concentration to the theoretical concentration, and compared with the detection method in the prior art that the trimethyl silanization derivative combined with the GC-MS method is a control group or an ICP-OES method for respectively detecting cyproconazole, dimethoate, chlorpyrimidol, benzopyrene, cadmium and mercury, the detection method in the invention has higher accuracy.
Example 3: verification of screening effect of detection result on risk of sexual precocity morbidity of children
To verify that the above detection method and the levels of 6 chemical toxins in the body can be used for early risk screening of precocious puberty in children, in this example, 17 urine samples (10 mL) of normal children and 20 urine samples (10 mL) of precocious puberty in children were collected, the samples were subjected to pretreatment of urine, and then mass spectrometry was performed for on-machine detection and data analysis, and the treatment method and detection conditions of the samples to be tested were exactly the same as those in example 2.
The data processing method in this embodiment: correlation between the pediatric precocious puberty onset profile and eight indicators was observed by calculating Spearman rank correlation analysis. Thermodynamic diagrams were drawn using Graphpad, and all statistical analyses were done in the statistical computing environment of SPSS (IBM, V26) software.
Fig. 7 shows the results of the detection of the urine samples of the normal control group and the premature infant group, wherein the detection results show that the content of the cyproconazole in the urine of the premature infant is 6.82 times higher than that of the normal infant (p < 0.001), the content of the dimethoate is 7.06 times higher than that of the normal infant (p < 0.001), the content of the benzopyrimidinol is 2.88 times higher than that of the normal infant (p < 0.001), the content of the cadmium is 2.86 times higher than that of the normal infant (p < 0.001), and the content of the mercury is 1.78 times higher than that of the normal infant (p < 0.001).
FIG. 8 is a graph showing correlation of the onset of precocity with cyproconazole, dimethoate, chloropyrinol, benzopyrene, cadmium and mercury, and a Spearman rank correlation analysis of the detection results shows that the onset of precocity with children in urine is positively correlated with the onset of precocity with cyproconazole, dimethoate, chloropyrinol, benzopyrene, cadmium and mercury, and the correlation coefficients are respectively: 0.86, 0.83 and 0.70, has statistical significance (see fig. 8). The results generally illustrate the residual situation of six endocrine disrupters, namely cyproconazole, dimethoate, chloropyrimidol, benzopyrene, cadmium and mercury, in urine of normal children and precocious puberty children, and can be used as a screening index of the precocious puberty risk of children.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.

Claims (9)

1. An endocrine disruptor for screening for a risk of sexual precocity in a child, comprising: one or more combinations of cyproconazole, dimethoate, chloropyrinol, benzopyrene, cadmium and mercury.
2. The endocrine disruptor for screening for a risk of sexual precocity in a child according to claim 1, wherein the sexual precocity in a child is caused by a chemical toxin in the environment.
3. Use of the endocrine disruptor of claim 1 in the preparation of a reagent or kit for screening for early childhood sexual precocity.
4. A kit for screening for early childhood sexual precocity comprising: the urine sample pretreatment reagent combination, eluent, mixed standard working solution, diluent, eluent/activating solution and compound solution; the urine sample pretreatment reagent combination comprises acetic acid-sodium acetate buffer solution, beta-glucuronidase/arylsulfatase and catechin.
5. The kit for screening early childhood sexual precocity according to claim 4, wherein the acetate-sodium acetate buffer, beta-glucosidase/arylsulfatase, catechin is used in an amount ratio of 2ml to 10 μl to 5mg; the acetic acid-sodium acetate buffer solution is formed by mixing 0.2M acetic acid aqueous solution and 0.2M sodium acetate aqueous solution according to the volume ratio of 1:6.
6. The kit for screening early childhood sexual precocity according to claim 4, wherein the mixed standard working fluid is an acetonitrile solution containing endocrine disrupters, the endocrine disrupters being one or more combinations of cyproconazole, dimethoate, chlorpyrinol, benzopyrene, cadmium and mercury, wherein the concentrations of cyproconazole, dimethoate, chlorpyrinol, cadmium and mercury are all 1 μg/mL, and the concentration of benzopyrene is 1ng/mL;
the diluent is an aqueous solution containing 2g/mL urea and 1g/mL sodium chloride;
the leaching liquid/activating liquid is water;
the eluent/activating solution is acetonitrile;
the complex solution is acetonitrile water solution containing 0.1% (v/v) formic acid, and the volume ratio of acetonitrile to water is 1:9.
7. The method of using the kit for screening early childhood sexual precocity of claim 4, comprising:
(1) Collecting urine samples of normal children and precocious puberty children, and treating the urine samples by using a urine sample pretreatment reagent combination;
(2) Preparing a mixed standard substance solution with a concentration gradient by using a mixed standard substance working solution in the kit;
(3) Performing LC-MS/MS mass spectrum sample injection on-machine detection on the prepared sample to be detected and the standard solution;
(4) And drawing a standard curve of the mixed standard substance solution according to the detection result, and screening the risk of the children sexual precocity morbidity.
8. The method of claim 7, wherein in step (3), the cyproconazole, dimethoate, chloropyrimidol and benzopyrene are detected by a liquid chromatography tandem mass spectrometer under the following mass spectrometry conditions:
the MRM parameters are:
9. the method of claim 7, wherein in step (3), the cadmium and mercury are analyzed by inductively coupled plasma mass spectrometry under the following conditions:
high frequency power: 1.2kW
Auxiliary gas flow rate: 1.1L/min
Sampling depth: 5.0mm
Plasma gas flow rate: 8.0L/min
Carrier gas flow rate: 0.7L/min
Mist room temperature: 5 DEG C
High frequency: 27.12MHz
Collision gas type: he (He)
Cell voltage: -21V
Collision gas flow rate: 6.0mL/min
Energy filter voltage: 5.0V.
CN202310191749.8A 2023-03-02 2023-03-02 Endocrine disrupter for screening sexual precocity risk of children and application thereof Pending CN116448901A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117805284A (en) * 2024-01-10 2024-04-02 裕菁科技(上海)有限公司 Method for simultaneously detecting multiple precocious biomarkers

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117805284A (en) * 2024-01-10 2024-04-02 裕菁科技(上海)有限公司 Method for simultaneously detecting multiple precocious biomarkers

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