CN109053497B - Preparation method and application of hydroxylated triclocarban - Google Patents
Preparation method and application of hydroxylated triclocarban Download PDFInfo
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- CN109053497B CN109053497B CN201810785477.3A CN201810785477A CN109053497B CN 109053497 B CN109053497 B CN 109053497B CN 201810785477 A CN201810785477 A CN 201810785477A CN 109053497 B CN109053497 B CN 109053497B
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Abstract
The invention discloses a preparation method and application of hydroxylated triclocarban, wherein the preparation method comprises the following steps: A. dissolving amino-chlorophenol and chlorobenzene isocyanate in a molar ratio of 1:0.8-1.2 in an organic solvent, and reacting at 45-65 ℃ for 10-30 h; B. pouring the reaction liquid into water, extracting with ethyl acetate, drying the organic phase, and removing the solvent under reduced pressure to obtain a crude product; C. purification by silica gel column chromatography gave hydroxylated triclocarban. The preparation method is simple, and the preparation conditions are not harsh; good universality to substrates and good product purity. The prepared hydroxylated triclocarban is used as a standard substance, can be used for qualitatively and quantitatively detecting the hydroxylated triclocarban in a biological sample by adopting an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer, has high detection sensitivity, low detection limit and accurate detection result, and lays a foundation for researching the migration transformation of the triclocarban in a biological body and the mechanism causing biological toxicity.
Description
Technical Field
The invention relates to the technical field of detection of hydroxyl metabolites of a broad-spectrum antibacterial agent triclocarban, and particularly relates to a preparation method and application of hydroxylated triclocarban.
Background
Triclocarban (TCC), the chemical name of which is 3,4,4 '-trichlorocarbanilide (3,4, 4' -trichlorocarbanilide), is a commonly used broad-spectrum antibacterial agent, and has been widely used in household and personal care products and medical supplies such as perfumed soaps, toothpastes, mouth wash, washing powders, deodorants, skin care products, beauty cosmetics, wound disinfectants, fabric antibacterial finishing agents and the like since the last 50 th century, wherein the TCC is added into the perfumed soaps in an amount of up to 1.5 wt%. TCC has been detected in humans in a variety of environmental media, wild animals and humans. In recent years, TCC has been identified as a novel environmental pollutant with the growing awareness of its environmental persistence, bioaccumulation, and biotoxicity. However, TCC is still produced in large quantities and widely used in the global range including China, and the human body exposure health risk is not completely clear.
The routes by which humans are exposed to TCC include direct skin contact of personal care products, as well as dietary, drinking, and dust contact. Due to the large use of personal care products in daily life, skin contact is considered to be the most prominent route by which humans are exposed to TCC. When a person takes a bath for 15min with a toilet soap containing 0.6 wt% of TCC, the soluble epoxide hydrolase in blood can be locally inhibited. Another important human exposure route is diet and drinking. Studies have shown that the daily intake of TCC by adults through the consumption of lettuce and spinach is 0.2-13 mug, and the daily intake through the consumption of domestic direct drinking water or tap water is 81.7ng and 425ng, respectively. To date, TCC has been commonly detected in the blood and urine of the population worldwide. In addition, TCCs may also be exposed to the fetus by the transfer of umbilical cord blood of pregnant women. Although TCCs have been produced and used for 60 years, the biotoxic effects of TCCs have not been gradually recognized and appreciated until the beginning of this century. TCC is an endocrine disruptor with significant reproductive and developmental toxicity to organisms. Recent results of human epidemiological investigations have shown that the TCC content in maternal plasma is significantly inversely correlated with gestational age of newborn infants, and that the level of TCC exposure in the mother has a correlation with fetal abnormalities.
After TCC enters a human body, most ingested TCC can generate hydroxylated I-phase oxidation metabolites under the action of cytochrome P450 enzyme (P450), mainly triclocarban 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC, which are monohydroxylated at the ortho-position or meta-position of a benzene ring, and in addition, a small amount of dihydroxylated triclocarban 2', 6-dioH-TCC and 3', 6-dioH-TCC can be generated. Further research proves that hydroxylated triclocarban (OH-TCC for short) in organisms can be continuously converted to generate quinonimine active intermediates with obvious toxic activity, and the active intermediates can be covalently combined with Glutathione (GSH) and macromolecular protein to bring great threat to human health. Therefore, in order to accurately reveal the human health risks to which TCC is exposed, the toxic effect of TCC hydroxyl metabolites on organisms and the molecular mechanism thereof need to be comprehensively considered; only based on the accurate quantification of the standard substance, the migration and transformation process and the toxicity mechanism of TCC in the organism can be comprehensively revealed. However, the biotoxicity effect of OH-TCC is generally ignored internationally at present, and reports of preparation methods of OH-TCC standard products are few, and the synthesis conditions are harsh, the separation and purification steps are complicated, and the yield is low.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a preparation method and application of hydroxylated triclocarban, and aims to solve the problems of complex preparation process, harsh preparation conditions, complicated separation and purification steps and low yield of the conventional hydroxylated triclocarban.
The technical scheme of the invention is as follows:
a method for preparing hydroxylated triclocarban, comprising:
step A, dissolving amino-chlorophenol and chlorobenzene isocyanate with a molar ratio of 1:0.8-1.2 in an organic solvent, and reacting at 45-65 ℃ for 10-30h to obtain a reaction mixed solution;
step B, pouring the reaction mixed solution into water, extracting with ethyl acetate, drying an organic phase with magnesium sulfate, and evaporating the solvent under reduced pressure to obtain a hydroxylated trichlorocarban crude product;
and step C, adding ethyl acetate and silica gel powder into the crude hydroxylated triclocarban product for redissolution, evaporating the solvent to dryness, purifying the crude hydroxylated triclocarban product by silica gel column chromatography with an eluent, and performing reduced pressure rotary evaporation and vacuum drying to obtain the hydroxylated triclocarban.
In the step A, the amino-chlorophenol is 2-ammonia-5-chlorophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated triclocarban obtained by reaction is as follows:
or the amino-chlorophenol is 2-chloro-5-aminophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-ammonia-4, 5-dichlorophenol, the chlorobenzene isocyanate is 4-chlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
the preparation method of the hydroxylated triclocarban, wherein in the step A, the organic solvent is N, N-dimethylformamide.
In the step C, the specification of the silica gel powder is 100-200 meshes, and the addition amount of the silica gel powder is 1.5-2.5 times of the sum of the mass of the amino-chlorophenol and the mass of the chlorobenzene isocyanate.
In the preparation method of the hydroxylated triclocarban, in the step C, when the purification is carried out by silica gel column chromatography, the inner diameter of the silica gel column is 26mm, and the filling height of the silica gel column is 100 mm.
In the preparation method of the hydroxylated triclocarban, the eluent is a mixed solvent of dichloromethane and ethyl acetate with a volume ratio of 20-30:1 in the step C.
A method for detecting hydroxylated triclocarban in a biological sample, wherein 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC are taken as target compounds; the hydroxylated triclocarban prepared by the preparation method is used as a standard substance, and an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer is used for detecting a target compound in a biological sample to be detected.
The chromatographic conditions detected by the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprise: using ACQUITY UPLC BEH C18 chromatographic column with specification of 1.7 μm, 100mm × 2.1mm, Waters; the mobile phase comprises acetonitrile and 2mM ammonium acetate water solution, and the flow rate of the mobile phase is 0.3 mL/min; carrying out chromatographic separation on each component in the biological sample to be detected by a gradient elution procedure according to different chromatographic retention time of each component in the biological sample to be detected under the conditions that the column temperature is 35 ℃ and the sample injection amount is 10 mu L;
the mass spectrum conditions detected by the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprise: the spraying voltage is 2600V, the temperature of an ion transmission tube is 350 ℃, the temperature of a sprayer is 300 ℃, mass spectrum cracking is carried out on each component after chromatographic separation in an electrospray negative ionization mode, scanning analysis is carried out on a monitoring ion pair of a target compound in a multi-reaction monitoring mode, and the target compound and the content of the target compound in a biological sample can be determined by comparing the target compound with a hydroxylated triclocarban standard product operated in parallel.
The method for detecting hydroxylated triclocarban in biological samples comprises the steps of taking 4.55min as the chromatographic retention time of 2'-OH-TCC and 6-OH-TCC and taking 4.12min as the chromatographic retention time of 3' -OH-TCC; monitor ion pairs for 2'-OH-TCC and 3' -OH-TCC at m/z328.9 → 142.0 and m/z328.9 → 168.0, and for 6-OH-TCC at m/z328.9 → 176.0 and m/z328.9 → 202.0; qualitative detection of hydroxylated triclocarban in biological samples.
The method for detecting hydroxylated triclocarban in a biological sample, wherein m/z328.9 → 168.0 is used as a monitoring ion pair for quantification of 2'-OH-TCC and 3' -OH-TCC, and m/z328.9 → 202.0 is used as a monitoring ion pair for quantification of 6-OH-TCC; carrying out quantitative detection on hydroxylated triclocarban in the biological sample by using the peak area of a chromatographic peak; and (5) calculating by a standard curve method to obtain a quantitative detection result.
Has the advantages that: the preparation process of the hydroxylated triclocarban is simple, the preparation conditions are not harsh, the used raw materials are low in price, the operation is easy, simple, convenient and quick, and the yield is high; the universality for the substrate is good, three isomers of the same type of hydroxylated triclocarban can be respectively prepared by selecting different substrates based on the same preparation, separation and purification route, and the purity of the obtained target compound is high. The hydroxylated triclocarban prepared by the method is used as a standard substance, can be used for qualitatively and quantitatively detecting the hydroxylated triclocarban in a biological sample, has high detection sensitivity, low detection limit and accurate detection result, and lays a foundation for researching the migration transformation of the triclocarban in a biological body and the mechanism causing biological toxicity.
Drawings
FIG. 1 shows a chromatogram-Mass spectrum (a) of the final product obtained in example 1 of the present invention measured in the Full Mass mode and a chromatogram-Mass spectrum (b) measured in the PRM mode.
FIG. 2 shows the NMR spectrum of the final product obtained in example 1 of the present invention.
FIG. 3 shows a chromatogram-Mass spectrum (a) of the final product obtained in example 2 of the present invention in the Full Mass mode and a chromatogram-Mass spectrum (b) in the PRM mode.
FIG. 4 is a NMR spectrum of the final product obtained in example 2 of the present invention.
FIG. 5 shows the chromatogram-Mass spectrum (a) of the final product obtained in example 3 of the present invention in the Full Mass mode and the chromatogram-Mass spectrum (b) in the PRM mode.
FIG. 6 shows the NMR spectrum of the final product obtained in example 3 of the present invention.
FIG. 7 is a chromatogram of three hydroxylated trichlorocarban isomers, 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC, in MRM mode in example 4 of this invention.
FIG. 8 is a chromatogram obtained in MRM mode of products resulting from TCC conversion in a biological sample according to the present invention.
Detailed Description
The invention provides a preparation method and application of hydroxylated triclocarban, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preparation method of hydroxylated triclocarban, which comprises the following steps:
step A, dissolving amino-chlorophenol and chlorobenzene isocyanate in a molar ratio of 1:0.8-1.2 (preferably in a molar ratio of 1:1) in an organic solvent (such as N, N-dimethylformamide), and reacting at a reaction temperature of 45-65 ℃ (preferably 50-60 ℃) for 10-30h (preferably 12-24 h) to obtain a reaction mixed solution;
step B, pouring the reaction mixed solution into water, extracting with ethyl acetate, drying an organic phase with magnesium sulfate, and evaporating the solvent under reduced pressure to obtain a hydroxylated trichlorocarban crude product;
and step C, adding ethyl acetate and silica gel powder into the crude hydroxylated triclocarban product for redissolution and evaporating the solvent to dryness, purifying the crude hydroxylated triclocarban product by silica gel column chromatography with an eluent (preferably a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 20-30:1 is used as the eluent) (when the purification is carried out by silica gel column chromatography, the inner diameter of the silica gel column is 26mm, the filling height of the silica gel column is 100mm), and carrying out reduced pressure rotary evaporation and vacuum drying to obtain the hydroxylated triclocarban.
Specifically, in the step a, the amino-chlorophenol is 2-ammonia-5-chlorophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated triclocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-chloro-5-aminophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-ammonia-4, 5-dichlorophenol, the chlorobenzene isocyanate is 4-chlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
the hydroxylated triclocarban is prepared by one step by taking amino-chlorophenol and chlorobenzene isocyanate as raw materials and realizing the process of generating substituted urea by reacting primary amine and isocyanate under specific conditions. The preparation method is simple, the preparation conditions are not harsh, the raw materials are cheap, the operation is easy, the method is simple, convenient and quick, the yield is high, the substrate universality is good, and the purity of the obtained target compound is high.
The invention also provides a method for detecting hydroxylated triclocarban in a biological sample, wherein 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC are used as target compounds; the hydroxylated triclocarban prepared by the preparation method is used as a standard substance, and an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer is used for detecting a target compound in a biological sample to be detected.
The chromatographic conditions detected by the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprise: using an ACQUITY UPLC BEH C18 chromatographic column (Waters) with a filler particle size of 1.7 μm and a column length × inner diameter of 100mm × 2.1 mm; the mobile phase is acetonitrile (component A) and 2mM ammonium acetate water solution (component B), and the flow rate of the mobile phase is 0.3 mL/min; carrying out chromatographic separation on each component in the biological sample to be detected by a gradient elution procedure according to different chromatographic retention time of each component in the biological sample to be detected under the conditions that the column temperature is 35 ℃ and the sample injection amount is 10 mu L;
the mass spectrum conditions detected by the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprise:spray voltage was 2600V, ion transport tube temperature was 350 deg.C, nebulizer temperature was 300 deg.C, in electrospray negative ionization mode (ESI)-) And performing mass spectrum cracking on each component after chromatographic separation, scanning and analyzing a monitoring ion pair of the target compound by a multi-reaction monitoring mode (MRM), and comparing the monitoring ion pair with a hydroxylated triclocarban standard product operated in parallel to determine the target compound and the content of the target compound in the biological sample.
Specifically, the method for detecting hydroxylated triclocarban in a biological sample comprises the following steps: 0-1min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 2:8, and the flow rate of the mobile phase is 0.3 mL/min; 2-5.5min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 8:2, and the flow rate of the mobile phase is 0.3 mL/min; 6-8min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 8:2, and the flow rate of the mobile phase is 0.3 mL/min.
Specifically, the method for detecting hydroxylated triclocarban in a biological sample comprises the steps of taking 4.55min as the chromatographic retention time of 2'-OH-TCC and 6-OH-TCC and taking 4.12min as the chromatographic retention time of 3' -OH-TCC; monitor ion pairs for 2'-OH-TCC and 3' -OH-TCC at m/z328.9 → 142.0 and m/z328.9 → 168.0, and for 6-OH-TCC at m/z328.9 → 176.0 and m/z328.9 → 202.0; qualitative detection of hydroxylated triclocarban in biological samples.
Specifically, the method for detecting hydroxylated triclocarban in a biological sample comprises a monitoring ion pair for quantification with m/z328.9 → 168.0 of 2'-OH-TCC and 3' -OH-TCC and a monitoring ion pair for quantification with m/z328.9 → 202.0 of 6-OH-TCC; carrying out quantitative detection on hydroxylated triclocarban in the biological sample by using the peak area of a chromatographic peak; and (5) calculating by a standard curve method to obtain a quantitative detection result.
The method for detecting the hydroxylated triclocarban in the biological sample uses the hydroxylated triclocarban prepared by the preparation method as a standard substance; the detection method can carry out qualitative detection and quantitative detection on the hydroxylated triclocarban in the biological sample, has high detection sensitivity, low detection limit and accurate detection result, and lays a foundation for researching the migration transformation of the triclocarban in organisms and the mechanism causing biological toxicity.
The present invention will be described in detail below with reference to examples.
Example 1
The preparation of 1- (4-chloro-2-hydroxyphenyl) -3- (3, 4-dichlorophenyl) urea (2 '-OH-TCC for short) and the reaction formula for synthesizing 2' -OH-TCC are as follows:
in a 50mL single-neck flask, 0.43g (3mmol) of 2-ammonia-5-chlorophenol and 0.56g (3mmol) of 3, 4-dichlorophenylisocyanate were put, dissolved in 15mL of DMF, and the reaction was stirred at 55 ℃ for 24 hours. The reaction mixture was poured into 30mL of water, extracted three times with 3X 50mL of ethyl acetate, the organic phase was dried over magnesium sulfate, the solvent was evaporated to dryness under reduced pressure, and then 10mL of ethyl acetate and 4mL of silica gel powder (100-mesh 200-mesh) were added for redissolution and evaporated to dryness. The crude product was purified by silica gel column chromatography with an inner diameter of 26mm and an effective height of 100mm packed with silica gel powder (100-200 mesh), using dichloromethane/ethyl acetate (20:1, v/v) as eluent, evaporating the eluent obtained after purification to dryness under reduced pressure and drying in vacuum to obtain 0.82g of solid final product with a yield of 82.8%.
The solid end product was dissolved in methanol and its structure and purity were characterized using Ultra Performance Liquid Chromatography (UPLC) -Q extraction combination quadrupole/orbitrap high resolution mass spectrometer (QE, Thermo Fisher Scientific, USA). UPLC ultra performance liquid chromatography system using an acquisition UPLC BEH C18 chromatography column (1.7 μm, 100mm × 2.1mm, Waters), column temperature: 35 ℃, flow rate: 0.30mL/min, sample size: 10 μ L. The mobile phase component A is acetonitrile, and the mobile phase component B is a water phase containing 2mM ammonium acetate; the elution gradient started at 20% A for 1.0min, reached 80% A at 2.0min and held for 6.0min, reached 100% A at 6.5min and held for 7.5min, and returned to 20% A at 8.0min and held for 2.0 min. QE mass spectrometry system using negative ionization mode in Full scan mode (Full MS) and parallel inversionShould monitor the mode (PRM) (to35Cl as an example), the structure of the final product is identified, the chromatogram-Mass spectrum of the final product measured in a Full Mass mode is shown in figure 1(a), the chromatogram-Mass spectrum of the final product measured in a PRM mode is shown in figure 1(b), the actual detection result of the final product conforms to the theoretical value of 2'-OH-TCC, the generation of high-purity 2' -OH-TCC is determined, and the chromatographic retention time is 4.92 min. The obtained final product was analyzed by NMR hydrogen spectroscopy using DMSO-d6 as a solvent, and the NMR hydrogen spectrum was shown in FIG. 2.1H-NMR (400MHz, DMSO-d 6): δ 10.55(s,1H),9.61(s,1H),8.28(s,1H),8.03(d,1H),7.88(d,1H),7.50(d,2H),7.27(dd,1H),6.85(d,1H),6.80(dd, 1H); the results show the purity of the 2' -OH-TCC prepared>99％。
Example 2
The preparation of 1- (4-chloro-3-hydroxyphenyl) -3- (3, 4-dichlorophenyl) urea (3 '-OH-TCC for short) and the reaction formula for synthesizing 3' -OH-TCC is as follows:
in a 50mL single-neck flask, 0.43g (3mmol) of 2-chloro-5-aminophenol and 0.56g (3mmol) of 3, 4-dichlorophenylisocyanate were put, dissolved in 15mL of DMF, and the reaction was stirred at 55 ℃ for 24 hours. The reaction mixture was poured into 30mL of water, extracted three times with 3X 50mL of ethyl acetate, the organic phase was dried over magnesium sulfate, the solvent was evaporated to dryness under reduced pressure, and 10mL of ethyl acetate and 4mL of silica gel powder (100 mesh, 200 mesh) were added for redissolution and evaporated to dryness. The crude product was purified by silica gel column chromatography with an inner diameter of 26mm and an effective height of 100mm packed with silica gel powder (100-200 mesh), using dichloromethane/ethyl acetate (20:1, v/v) as eluent, and the eluent obtained after purification was evaporated to dryness under reduced pressure and dried under vacuum to obtain 0.78g of solid final product with a yield of 78.8%.
The solid end product is dissolved in methanol, and the specific parameters and steps for detection and analysis are the same as in example 1, and will not be described herein. The chromatogram-Mass spectrum of the final product is shown in FIG. 3(a) in Full Mass mode, and the chromatogram-Mass spectrum in PRM mode is shown in FIG. 3(b), and its actual detection is performedThe results agree with the theoretical values of 3'-OH-TCC and confirm the formation of 3' -OH-TCC with a chromatographic retention time of 4.32 min. The final product was analyzed by NMR spectroscopy using DMSO-d6 as a solvent, and the NMR spectrum obtained is shown in FIG. 4.1H-NMR (400MHz, DMSO-d 6): δ 10.12(s,1H),8.92(s,1H),8.83(s,1H),7.86(d,1H),7.50(d,1H),7.30(dd,2H),7.28(d,1H),7.18(d,1H),6.81(dd, 1H); the results show the purity of the prepared 3' -OH-TCC>99％。
Example 3
1- (4-chlorphenyl) -3- (4, 5-dichloro-2-hydroxyphenyl) urea (short for 6-OH-TCC) is prepared, and the reaction formula for synthesizing the 6-OH-TCC is as follows:
in a 50mL single-neck flask were charged 0.43g (3mmol) of 2-amino-4, 5-dichlorophenol and 0.56g (3mmol) of 4-chlorobenzeneisocyanate, and the mixture was dissolved in 15mL of DMF and reacted at 55 ℃ for 24 hours with stirring. The reaction mixture was poured into 30mL of water, extracted three times with 3X 50mL of ethyl acetate, the organic phase was dried over magnesium sulfate, the solvent was evaporated to dryness under reduced pressure, and 10mL of ethyl acetate and 4mL of silica gel powder (100 mesh, 200 mesh) were added for redissolution and evaporated to dryness. The crude product was purified by silica gel column chromatography with an inner diameter of 26mm and an effective height of 100mm packed with silica gel powder (100-200 mesh), using dichloromethane/ethyl acetate (30:1, v/v) as eluent, and the eluent obtained after purification was evaporated to dryness under reduced pressure and dried under vacuum to obtain 0.86g of solid final product with a yield of 86.9%.
The solid end product is dissolved in methanol, and the specific parameters and steps for detection and analysis are the same as in example 1, and will not be described herein. The chromatogram-Mass spectrum of the final product is shown in FIG. 5(a) in the Full Mass mode, the chromatogram-Mass spectrum is shown in FIG. 5(b) in the PRM mode, the actual detection result is consistent with the theoretical value of 6-OH-TCC, the generation of high-purity 6-OH-TCC is determined, and the chromatogram retention time is 4.92 min. The final product was analyzed by NMR using DMSO-d6 as a solvent, and the NMR spectrum was shown in FIG. 6.1H-NMR (400MHz, DMSO-d 6): δ 10.81(s,1H),9.52(s,1H),8.40(s,1H),8.32(s,1H),7.46(d,2H),7.32(d,2H),6.99(s, 1H); the results show the purity of the prepared 6-OH-TCC>99％。
EXAMPLE 4 detection of hydroxylated Trichlorocarban (abbreviated as OH-TCC) in biological samples
(1) Optimizing the relevant detection parameters of the hydroxylated triclocarban prepared by the invention
A UPLC-TSQ triple quadrupole tandem mass spectrometer (TSQ, Thermo Fisher Scientific, USA) was used to establish the detection method for OH-TCC. UPLC ultra performance liquid chromatography system using an acquisition UPLC BEH C18 chromatography column (1.7 μm, 100mm × 2.1mm, Waters), column temperature: 35 ℃, flow rate: 0.30mL/min, sample size: 10 μ L. Mobile phase component a was acetonitrile and mobile phase component B was an aqueous phase containing 2mM ammonium acetate, and the gradient elution procedure is shown in table 1. TSQ mass spectrometry system using electrospray negative ionization mode (ESI)-) Multiple reaction monitoring mode (MRM) scans were selected, spray voltage 2600V, ion transfer tube temperature 350 ℃, nebulizer temperature 300 ℃.
TABLE 1 gradient elution procedure
Note: a (%) and B (%) are A, B volume percent of the two components.
The three isomers of the hydroxylated triclocarban prepared by the invention, namely 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC, are subjected to mass spectrum condition optimization by using a UPLC-TSQ triple quadrupole tandem mass spectrometer, and chromatograms of the 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC in an MRM mode are shown in FIG. 7; the parameters of the mass spectrum for detecting the hydroxylated triclocarban isomer through optimization are shown in table 2. Among them, 2'-OH-TCC and 3' -OH-TCC have monitoring ion pairs of m/z328.9 → 142.0 and m/z328.9 → 168.0, and since the chromatographic peaks of the two substances are completely separated, 328.9 → 168.0 with higher response is selected as the monitoring ion pair for quantification. The chromatographic retention time of 6-OH-TCC is the same as 2' -OH-TCC and separation can be performed by specific ion fragments, which monitor the ion pair as m/z328.9 → 176.0 and m/z328.9 → 202.0. Among them, 328.9 → 202.0 with higher response was selected as the quantitative monitoring ion pair for 6-OH-TCC.
TABLE 2 correlation of the parameters measured for the three isomers of triclocarban
Note:athe daughter ions were quantified.
(2) Preparing a biological sample
Human normal hepatocytes L02 in logarithmic growth phase were selected and exposed TCC was added to the cell culture medium. After 48h exposure, the cell culture was removed, the cells were washed 3 times with Phosphate Buffered Saline (PBS), trypsinized and centrifuged (1000rpm, 5 min); removing matrix, adding pre-cooled 80% methanol/water extractive solution, repeatedly freezing and thawing cells for 5 times with liquid nitrogen, and centrifuging at low temperature and high speed (15,000rpm, 10min,4 deg.C) to obtain cell extractive solution to be detected, i.e. biological sample to be detected.
(3) Detection of hydroxylated triclocarban in biological samples
Three isomers of hydroxylated triclocarban, namely 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC, are taken as standard substances, and the analysis and detection conditions are the same as those in the embodiment (1), and the chromatogram measured in the MRM mode is shown in FIG. 8, wherein the analysis and detection conditions are the same as those in the embodiment (1). The results show that in biological samples, OH-TCC was detected and that metabolites predominated on 2' -OH-TCC followed by 6-OH-TCC. Quantitatively determining the content of 2' -OH-TCC in the cell extract to be 0.916 nmol.L–1The content of 3' -OH-TCC is 0.058 nmol.L–1The content of 6-OH-TCC is 0.632 nmol.L–1This indicates that the TCC in the cell has an oxidative metabolic reaction.
In conclusion, the preparation method and the application of the hydroxylated triclocarban are provided by the invention, the preparation method of the hydroxylated triclocarban is simple, the preparation conditions are not harsh, the used raw materials are cheap, the operation is easy, the method is simple and fast, and the yield is high; the universality for the substrate is good, three isomers of the same type of hydroxylated triclocarban can be respectively prepared by selecting different substrates based on the same preparation, separation and purification route, and the purity of the obtained target compound is high. The hydroxylated triclocarban prepared by the method is used as a standard substance, can be used for qualitative detection and quantitative detection of hydroxylated triclocarban in a biological sample, has high sensitivity and accurate detection result, and lays a foundation for researching the migration transformation of triclocarban in organisms and the mechanism causing biological toxicity.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (2)
1. A preparation method of hydroxylated triclocarban is characterized by comprising the following steps:
a, dissolving amino-chlorophenol and chlorobenzene isocyanate in a molar ratio of 1:1 in an organic solvent, and reacting at 55 ℃ for 24 hours to obtain a reaction mixed solution;
step B, pouring the reaction mixed solution into water, extracting with ethyl acetate for three times, drying an organic phase with magnesium sulfate, and evaporating the solvent under reduced pressure to obtain a hydroxylated trichlorocarban crude product;
step C, adding 10mL of ethyl acetate and 4mL of silica gel powder into the crude hydroxylated triclocarban product for redissolving and evaporating the solvent to dryness, purifying the crude hydroxylated triclocarban product by silica gel column chromatography with an eluent, and performing reduced pressure rotary evaporation and vacuum drying to obtain the hydroxylated triclocarban;
in the step C, the specification of the silica gel powder is 100-200 meshes; when purifying through silica gel column chromatography, the inner diameter of the silica gel column is 26mm, and the filling height of the silica gel column is 100 mm; the eluent is a mixed solvent of dichloromethane and ethyl acetate with the volume ratio of 20-30: 1;
in the step A, the amino-chlorophenol is 2-ammonia-5-chlorophenol, and the chlorobenzene isThe isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-chloro-5-aminophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-ammonia-4, 5-dichlorophenol, the chlorobenzene isocyanate is 4-chlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
the organic solvent is N, N-dimethylformamide;
the hydroxylated triclocarban is used for qualitative and quantitative detection of hydroxylated triclocarban in biological samples.
2. A method for detecting hydroxylated triclocarban in a biological sample is characterized in that 2'-OH-TCC, 3' -OH-TCC and 6-OH-TCC in the biological sample are taken as target compounds; detecting a target compound in a biological sample to be detected by using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer by taking hydroxylated triclocarban as a standard substance;
the preparation method of the hydroxylated triclocarban serving as a standard comprises the following steps:
a, dissolving amino-chlorophenol and chlorobenzene isocyanate in a molar ratio of 1:1 in an organic solvent, and reacting at 55 ℃ for 24 hours to obtain a reaction mixed solution;
step B, pouring the reaction mixed solution into water, extracting with ethyl acetate, drying an organic phase with magnesium sulfate, and evaporating the solvent under reduced pressure to obtain a hydroxylated trichlorocarban crude product;
step C, adding ethyl acetate and silica gel powder into the crude hydroxylated triclocarban product for redissolution and evaporating the solvent to dryness, purifying the crude hydroxylated triclocarban product by silica gel column chromatography with an eluent, and performing reduced pressure rotary evaporation and vacuum drying to obtain the hydroxylated triclocarban;
in the step C, the specification of the silica gel powder is 100-200 meshes, and the addition amount of the silica gel powder is 1.5-2.5 times of the mass sum of the amino-chlorophenol and the chlorobenzene isocyanate as raw materials during redissolution; when purifying through silica gel column chromatography, the inner diameter of the silica gel column is 26mm, and the filling height of the silica gel column is 100 mm; the eluent is a mixed solvent of dichloromethane and ethyl acetate with the volume ratio of 20-30: 1;
in the step A, the amino-chlorophenol is 2-ammonia-5-chlorophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated triclocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-chloro-5-aminophenol, the chlorobenzene isocyanate is 3, 4-dichlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
or the amino-chlorophenol is 2-ammonia-4, 5-dichlorophenol, the chlorobenzene isocyanate is 4-chlorobenzene isocyanate, and the structural formula of the hydroxylated trichlorocarban obtained by the reaction is shown as follows:
taking 4.55min as chromatographic retention time of 2'-OH-TCC and 6-OH-TCC as standard samples, and taking 4.12min as chromatographic retention time of 3' -OH-TCC as standard samples; the monitoring ion pair of 2'-OH-TCC and 3' -OH-TCC as standard substances is represented by m/z328.9 → 142.0 and m/z328.9 → 168.0, and the monitoring ion pair of 6-OH-TCC as standard substances is represented by m/z328.9 → 176.0 and m/z328.9 → 202.0; carrying out qualitative detection on hydroxylated triclocarban in a biological sample;
m/z328.9 → 168.0 is used as a monitoring ion pair for quantifying 2'-OH-TCC and 3' -OH-TCC of the standard, and m/z328.9 → 202.0 is used as a monitoring ion pair for quantifying 6-OH-TCC of the standard; carrying out quantitative detection on hydroxylated triclocarban in the biological sample by using the peak area of a chromatographic peak; calculating by a standard curve method to obtain a quantitative detection result;
the chromatographic conditions of the assay include: using ACQUITYUPLCBEHC18 chromatographic column with specification of 1.7 μm, 100mm × 2.1mm, Waters; the mobile phase comprises acetonitrile and 2mM ammonium acetate water solution, and the flow rate of the mobile phase is 0.3 mL/min; carrying out chromatographic separation on each component in the biological sample to be detected by a gradient elution procedure according to different chromatographic retention time of each component in the biological sample to be detected under the conditions that the column temperature is 35 ℃ and the sample injection amount is 10 mu L;
the mass spectrometric conditions of the detection include: the spraying voltage is 2600V, the temperature of an ion transmission tube is 350 ℃, the temperature of a sprayer is 300 ℃, mass spectrum cracking is carried out on each component after chromatographic separation in an electrospray negative ionization mode, scanning analysis is carried out on a monitoring ion pair of a target compound in a multi-reaction monitoring mode, and the target compound and the content of the target compound in a biological sample can be determined by comparing the target compound with a hydroxylated triclocarban standard product operated in parallel;
the gradient elution procedure was: 0-1min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 2:8, and the flow rate of the mobile phase is 0.3 mL/min; 2-5.5min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 8:2, and the flow rate of the mobile phase is 0.3 mL/min; 6-8min, wherein the mobile phase is a mixture of acetonitrile and 2mM ammonium acetate water solution in a volume ratio of 8:2, and the flow rate of the mobile phase is 0.3 mL/min.
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