Effect-directed identification of targeted and non-targeted androgen disruptors

Abstract

Implementations herein relates to effect-directed identification of targeted and non-targeted androgen disruptors. The implementations include primary separation, androgenic activity testing, high throughput separation and preparation of toxicants, active component scanning based on high performance liquid chromatography-time of flight mass spectrometry, targeted screening of suspicious androgenic substances, non-target identification of androgenic compounds combining mass spectrum, chromatography and toxicity characteristics and toxicity confirmation. The implementations perform separation using SPE and preparative separation in series to obtain high throughput separation fractions, utilize DMSO as a protective agent to optimize concentration of second fractions, utilize target databases to achieve target identification of key toxicants. In addition, assess mass spectrum characteristic identification based on TOF-MS library spectrum, assess chromatography characteristic identification methods based on relationship between retention times and characteristics of compounds, assess toxicity characteristic identification methods based on molecular dynamics simulation techniques, and further assess non-targeted identification and determination of key androgenic substances in the fractions.

Claims

1. A method for effect-directed identification of targeted or non-targeted androgen disruptors, the method comprising: (1) extracting organic substances of solid samples using accelerated solvent extraction after the samples are dried and milled or centrifuging or filtering liquid samples and performing enrichment of pollutants with different polarities using solid phase extraction columns in series; (2) performing primary separation by elution using organic solvents with different hydrophibicities on solid phase extraction columns in series; (3) testing androgenic or anti-androgenic activity using stably transfected MDA-Kb2 cell line; (4) performing high throughput separation and preparation of androgenic or anti-androgenic fractions, performing fractionation on the fractions using preparative chromatography based on polarities of substances or using gel exclusion chromatography based on molecular sizes, adding DMSO as protection liquid and concentrating by nitrogen blowing to obtain secondary fractions; (5) testing the androgenic or anti-androgenic activity of the secondary fractions using the MDA-Kb2 cell line; (6) scanning and analyzing the secondary fractions with detectable activities to obtain primary and secondary mass spectrum using high performance liquid chromatography-time of flight mass spectrometry; (7) establishing a database for suspicious substances associated with androgenic activity, importing the database of target toxicants, calculating accurate mass and performing fragment analysis on target substances of secondary fractions, and performing targeted screening by comparison primary and secondary mass spectrum of target substances; (8) performing non-targeted screening of androgenic fractions. Identifying non-target androgenic compounds based on mass characteristics, chromatographic characteristics and toxicity characteristics. (9) obtaining standards for quantitative analysis and measuring EC50, calculating toxicity contribution of toxicants, extracting and confirming the toxicants by configuring blank media based on concentrations obtained from the quantitative analysis of the toxicants.

2. The method of claim 1, wherein the organic solvents for the accelerated solvent extraction in step (1) contains a mixed solvent of dichloromethane and n-hexane and a solvent of methanol, a volume ratio of dichloromethane and n-hexane is 1:1, the samples are extracted twice in series and collected separately, and amino columns and HLB columns in series solid phase extraction of the liquid samples is used to achieve enrichment of pollutants with different polarities.

3. The method of claim 1, wherein enriched liquid of the solid samples in step (2) is separated using amino columns and HLB columns in series, liquid samples are separated using HLB columns, the amino columns are eluted with a mixture of acetonitrile and toluene with a volume ratio of 3:1, substances enriched on HLB columns are eluted with 10 mL mixed solvents of methanol and dichloromethane with volume ratio of 1:1, 10 mL mixed solvents of dichloromethane and n-hexane with volume ratio of 4:1, and 10 mL solvent of hexane in series.

4. The method of claim 1, wherein parameters of the high throughput separation and preparation comprise: gel permeation chromatography: J2 Scientific, AccuPrep MPS, materials of column packing: Bio-Beads S-X3, an inside diameter of: 3 cm, a length: 20 cm, eluent: ethyl acetate and cyclohexane with volume ratio of 1:1, a flow rate: 5 mL/min; preparation chromatograph: Waters AutoPurification HPLC, column: Waters XBridge C18 preparative column, preparative column dimensions of 19 mm×150 mm, a particle diameter of 5 μm, a flow rate: 5 mL/min, and a mobile phase: methanol and water.

5. The method of claim 4, wherein the protection liquid in step (4) is dimethyl sulfoxide (DMSO) to optimize concentration of preparative fractions. A certain amount of DMSO is added at a volume ratio of 20% of the final volume.

6. The method of claim 1, wherein instrument parameters used in step (6) comprise: HPLC: Agilent1260, column: Agilent C18 columns, a column size is 2.1 mm×150 mm, a particle diameter of 2.5 μm, a column temperature: 30° C., a mobile phase: Acetonitrile, a volume percentage of 5% acetonitrile in water; a flow rate: 300 μL/min, MS: Triple TOF 5600-AB ACIEX, Ion source: ESI, Ionization mode: positive ion mode and negative mode; MS scan range: 100-2000 m/z, MSMS scan range: 60-1250 m/z, cluster voltage: ±80V, and Collision voltage: ±35±15 eV.

7. The method of claim 1, wherein the software in step (7) is PeakView, XIC Manager is used for importing of the database of target toxicants, the database of the target toxicants comprises molecular formulas, ionization and parent mass parameters, and the method further comprises: setting peak intensity Intensity>1000, signal to noise ratio S/N>10 and isotope Cl, S, P parameters, extracting primary mass spectrum which is consistent with those in target databases determining whether the substance is the target substance based on a determination of matching between the secondary mass spectrum corresponding to the extracted peak and the secondary mass spectrum of the target substances that have been reported in literatures and contain at least two characteristics of secondary ions.

8. The method of claim 1, wherein the primary spectrum in step (8) is obtained using PeakView by retrieving accurate m/z of mass peaks via ion extraction window XIC, and the method further comprises: obtaining the secondary spectrum of the substance using Information Dependent Acquisition, obtaining a list of suspicious compounds by comparing chemical compounds from Chemspider and the primary and secondary mass spectrum, and wherein the structures: are further filtered based on chromatographic characteristics obtained from relationship between retention times and molecular weight ranges of individual fractions or relationship between retention times and Log Kow of the individual compounds: Log Kow=0.11501T.sub.R-0.21618, r2=0.9769, and are further predicted as androgenic or anti-androgenic by using toxicity characteristic, performing molecular dynamics simulation for linkable substances, monitoring whether H12 relocation is stable within 20 ns; and determining the substance as suspected toxicants if the H12 relocation is stable within 20 ns.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The detailed description is described with reference to the accompanying FIG.s. In the FIG.s, the left-most digit(s) of a reference number identifies the FIG. in which the reference number first appears. The same reference numbers in different FIG.s indicate similar or identical items.

[0031] FIG. 1 is a diagram illustrating validation of recovery rates for an optimized method for concentrated preparation of fractions.

[0032] FIG. 2 is a diagram showing toxicity test results of primary fractions separated from sensitive areas of soil samples of Chemical Industrial Park.

[0033] FIG. 3 is a diagram showing toxicity test results of preparative fractions of soil samples.

[0034] FIG. 4 is a diagram of primary and secondary spectrum showing results of targeted screening of Phthalic anhydride and indicating the presence of phthalic anhydride in the samples.

[0035] FIG. 5 is a diagram of primary and secondary spectrum showing target screening results of cyclohexylamine and indicating the presence of cyclohexylamine.

[0036] FIG. 6 is a diagram illustrating prediction of anti-androgen toxicity caused by potential toxic structures.

DETAILED DESCRIPTION

[0037] To further understand the present disclosure, the accompanying drawings and the implementations of the present disclosure will be described in detail.

[0038] The implementations relate to effect-directed identification of targeted and non-targeted androgen disruptors. The implementations may include the following operations.

[0039] (1) Extraction of samples: Organic substances in solid samples were extracted following accelerated solvent extraction using Dionex ASE350. The extraction process includes extraction using mixed solvents of dichloromethane and n-hexane (1:1, v:v) for three times and extraction using pure methanol solvents for three times, collected separately.

[0040] (2) Solid Phase Extraction: The solid phase extraction columns are activated using 10 mL hexane, 10 mL of dichloromethane, and 10 mL of methanol, extracts from accelerated solvent extraction are further enriched and purified using solid phase extraction columns, and flow is controlled at 1-2 drops/sec.

[0041] (3) Eluting and concentrating: sequentially using 10 mL of mixed solution including methanol: Dichloromethane=1:1 (volume ratio), 10 mL of mixed solvents including dichloromethane: N-hexane=4:1 (volume ratio), and 10 mL of hexane to elute substances form the HLB columns for collection. After rotary evaporation and nitrogen blowing, eluent was concentrated in sample vials and volumes were determined. Depending on the eluent, extracted organic substances were divided into four fractions, which were named as 0, 1, 2 and 3 based on hydrophibicity varieties.

[0042] (4) Reporter gene testing using MDA-Kb2 cell line: Concentrates of the primary fractions are solvent-replaced by dimethyl sulfoxide (DMSO), and are further diluted to seven different gradients with three parallels. Reporter gene testing using MDA-Kb2 cell is used to detect androgenic and/or anti-androgenic activity. FIG. 2 is a diagram showing primary toxicity test results of fractions primarily separated.

[0043] (5) High throughput separation and preparation: Preparative chromatography techniques are used to effectively perform high-throughput separation of the primary fractions based on polarities and retention times of fractions. Its parameters are as follows: preparation chromatograph: Waters AutoPurification HPLC; column: Waters preparative column Waters XBridge C18 preparative columns, preparative column dimensions of 19 mm×150 mm, a particle diameter of 5 μm, a flow rate: 5 mL/min; a mobile phase: Methanol, water; Collection methods: 15 mL of glasses are used to collect according to the time period.

[0044] Mobile phase gradient is provided as follow:

TABLE-US-00004 Methanol Percent Water Time (min) percentage (%) Content (%) 0 50 50 50 100 0 65 100 0

[0045] Collection methods are provided as follows:

TABLE-US-00005 Collection Fraction time (min) 1 0.5 2 1 3 1.5 4 2 5 2.4 6 3 7 3.5 8 4 9 4.5 10 5 11 5.7 12 6.2 13 6.7 14 7.2 15 7.7 16 8.2 17 8.7 18 9.2 19 9.7 20 10.2 21 11.4 22 12.6 23 13 24 14 25 15 26 16 27 17 28 18 29 19 30 20 31 21 32 22 33 23 34 24 35 25 36 26 37 27 38 28 39 29 40 30 41 31 42 32 43 33 44 34 45 36 46 38 47 40 48 42 49 45 50 50 51 65

[0046] (6) Concentrating method for fractionation: During the concentration, the implementations include DMSO as a protection agent to improve recovery rates. Specifically, optimized concentration method is as follows:

TABLE-US-00006 Secondary Fraction Concentration method  1-44 After adding protective agent DMSO, directly nitrogen blowing, Volume 45-51 DMSO was added as protective agent after collecting fractions together that belong to each other, performing rotary evaporation and determining volumes.

[0047] (7) Separating secondary fractions and reporter gene testing using MDA-Kb2 cell line: A portion of the concentrates of the secondary fraction is obtained, diluted into seven gradients with three parallels for further reporter gene testing based on MDA-Kb2 cell to detect proposed anti-androgenic activity. With standard anti-androgen flutamide for comparison, Graphpad Software for fitting, IC20 of fractions is achieved to calculate TEQ of each fraction. FIG. 3 is a diagram showing toxicity test results from separated fractions prepared using soil sampling points.

[0048] (8) Qualitative identification of compounds in secondary fraction 23 of primary fraction 2: Performing further analysis on anti-androgenic secondary fraction 23 based on high performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF), obtaining primary and secondary mass spectrum; and performing comparison through database of the instrument and an online database (e. g., Chemspider) to obtain a list of suspicious compounds. Instrument parameters used in step (8) includes: HPLC: Agilent 1260; column: Agilent C18 column, Size of 2.1 mm×150 mm, a particle diameter of 2.5 μm; a column temperature: 30° C.; a mobile phase: Acetonitrile, a volume percentage of 5% acetonitrile in water; a flow rate: 300 μL/min; and Mobile phase gradient is provided as follow:

TABLE-US-00007 Percent Water Acetonitrile Time (min) Content (%) percentage (%) 0 100 0 1 100 0 15 50 50 25 0 100 30 0 100 30.5 100 0 40 100 0

[0049] MS: Triple TOF 5600-AB ACIEX; Ion source: ESI; Ionization mode: Positive mode and negative mode; MS scan range: 100-2000 m/z; MSMS scan range: 60-1250 m/z; To cluster voltage: ±80V; Collision voltage: ±35±15 eV.

[0050] (9) Targeted screening of suspicious androgenic substances in secondary fraction 23: It has been reported that 21 types of androgenic substances and 124 types of anti-androgenic substances proposed in databases. The database includes chemical names, CAS number, information related to EC20, EC50, typical characteristic ions, Log Kow associated with androgenic and/or anti-androgenic substances. Further information is also available such as 235 kinds of chemical raw materials and 166 kinds of related chemical products that are registered in Chemical Industrial Park. The registered information includes chemical names, CAS number, information related to EC20, EC50, typical characteristic ions, Log Kow associated with substances registered in Chemical Industrial Park. Mass spectral data analysis software PeakView was used, XIC Manager was used to import target substance databases including characteristics of ion pairs of androgenic and/or anti-androgenic substances and databases including basic physical and chemical information of chemicals registered in Chemical Industry Park. The method further includes: setting peak intensity Intensity>1000, signal to noise ratio S/N>10 and isotope Cl, S, P. Extract primary mass spectrum which are consistent with those in target databases. Followed by literatures or prediction of molecular structure fracture modes, the implementations determines whether the substance is the target substance based on a determination of matching between the secondary mass spectrum corresponding to the extracted peak and the secondary mass spectrum of the target substance that has been reported in literatures and contains at least two characteristics of secondary ions. Targeted screening results are shown in FIG. 4 and FIG. 5.

[0051] (10) Non-targeted screening of suspicious androgenic substances in secondary component 23: The primary spectrum is obtained using PeakView by retrieving accurate m/z of mass peaks via ion extraction window XIC, by 7 golden rules Namely by limiting types of elements, valence of restricted substances, unsaturation, and other basic chemical information to obtain molecular formula: C16H35N, obtaining the secondary spectrum of the substance using Information Dependent Acquisition, obtaining a list of 26 suspicious compounds by comparing chemical compounds from Chemspider and the primary and secondary mass spectrum, based on a relationship between preparative chromatography retention time and polarity of compounds: Log Kow=0.11501tR-0.21618, calculating Log Kow ranges of the substance corresponding to secondary component 23:1.16-1.28, screening in the list of suspicious compounds based on hydrophbicity ranges thereof, further screening based on secondary spectrum and those in literatures sharing at least two characteristics of product ions. 4 compounds were screened from 26 possible molecular structures. Screening results are provided as follows:

TABLE-US-00008 CAS category Chinese name English name number Non- DI-SEC-OCTYLAMINE 5412-92-0 targeted Hexadecylamine 1-Hexadecylamine 143-27-1 Di-iso- Bis(2-ethylhexyl)amine 106-20-7 octylamine Di-n-octylamine Dioctylamine 1120-48-5 targeted Phthalic Phthalic anhydride 85-44-9 anhydride Cyclohexylamine Cyclohexylamine 108-91-8

[0052] Finally, the toxicity characteristic is determined. Chemoffice is used to construct ligands and receptors of 3D structures, structures of the ligands are optimized, and a Surflex-Dock module of Sybyl software is linked to an AR-LBD active site of a small molecule for a test, and then the implementations use Gromacs4.0 molecular modeling software for MD simulations adopting for field processing CHARMM27 protein receptors and ligand molecules. TIP3P based spherical layers of water molecules are added to every composite system. A minimum spacing edge of a solute and solvent is 10 Å. Sodium or chloride ion is added so that the system is in equilibrium charge state. All systems are used a steepest-descent method to optimize energy, and then to restrict ligand positions, within 40 picoseconds (PS) time the temperature rises from 0K to 300K, in the condition of one atmosphere and 300K, balancing 1 nanosecond (ns) and molecular dynamics simulations are followed, wherein the electrical interaction is applied with a particle mesh Ewald (PME) method to calculate, Van der Waals cutoff is set to 10 Å, all simulations for 20 ns, and a step is set to 2 femtoseconds (fs), saving every 2 ps. Molecular dynamics simulation data obtained are GROMACS4.0 for processing, wherein H12 of phthalic anhydride and di-n-octylamine is stable within 20 ns, determining that the substances are anti-androgenic. The molecular dynamics simulation results are shown in FIG. 6.

[0053] (11) Confirmation of toxicants: Standards of identified toxicants were purchased, and high performance liquid chromatography tandem mass spectrometry (LC-MSMS) was used for quantification. Quantitative results are provided as follows, and the implementations further include configuring original blank media based on a concentration obtained from the quantitative analysis of the toxicants, and calculating toxicity contribution of the toxicants.

TABLE-US-00009 Sampling points Possible S4 S6 S8 S9 S11 S13 sources Phthalic 132 114 471 1158 701 7 production anhydride material (μg/kg soil) Di-n- ND ND ND ND 137 ND Amine octylamine solvents, (μg/kg intermediates soil) and extraction agent

CONCLUSION

[0054] Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of implementing the claims.