CN108918713B - Ammonium triclosan sulfonate and preparation method and application thereof - Google Patents

Ammonium triclosan sulfonate and preparation method and application thereof Download PDF

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CN108918713B
CN108918713B CN201810791720.2A CN201810791720A CN108918713B CN 108918713 B CN108918713 B CN 108918713B CN 201810791720 A CN201810791720 A CN 201810791720A CN 108918713 B CN108918713 B CN 108918713B
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triclosan
sulfonic acid
sulfonate
ammonium
methanol
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CN108918713A (en
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蔡宗苇
张宏娜
宋媛媛
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Hong Kong Baptist University HKBU
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    • 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
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    • 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
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    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • 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
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Abstract

The invention discloses ammonium triclosan sulfonate and a preparation method and application thereof, wherein the structural formula of the ammonium triclosan sulfonate is shown in the specification
Figure DDA0001735073670000011
The ammonium triclosan sulfonate can stably exist in a solid state, replaces triclosan sulfonic acid as a standard substance for detecting the triclosan sulfonic acid in a biological sample, can meet the requirement of accurate quantitative detection, has high detection sensitivity, and lays a foundation for researching the migration and transformation of triclosan in a living body and the mechanism causing biological toxicity.

Description

Ammonium triclosan sulfonate and preparation method and application thereof
Technical Field
The invention relates to the technical field of antibacterial agent detection, and particularly relates to ammonium triclosan sulfonate and a preparation method and application thereof.
Background
Triclosan (TCS), known by the chemical name 5-chloro-2- (2,4-dichlorophenoxy) phenol (5-chloro-2- (2,4-dichlorophenoxy) phenol), is a broad-spectrum, highly effective antimicrobial agent, and has been widely added to personal care products such as toothpaste, soap, shampoo, skin cream, washing liquid, etc., and household daily necessities, with a content of 0.1-0.3 wt%, since the 70 s of the 20 th century. Due to its wide application range and large dosage, triclosan has been detected in a variety of environmental media and organisms worldwide.
The human body may expose triclosan by ingestion and skin contact. Triclosan is detected in urine, serum, plasma and breast milk of a human body; in addition, triclosan in the maternal serum can also cross the placental barrier and be exposed to the fetus via the umbilical cord blood. With the increasing depth of related research, the biotoxicity effect of triclosan is gradually recognized. Potential toxic effects of triclosan include endocrine disruption, hepatotoxicity, genotoxicity, impaired thyroid function, developmental disorders, oxidative stress, and the like. After triclosan enters a human body, metabolic reactions can occur simultaneously, lipophilic triclosan is converted into phase II metabolite triclosan sulfonic acid (TCSS) and triclosan glucuronic acid (TCSG) with stronger polarity and better water solubility mainly through sulfonation reactions and glucuronidation reactions, and then the triclosan is discharged out of the body through urine and feces. Therefore, the research on the metabolic process of the triclosan in the organism is of great significance for the comprehensive evaluation of the biological toxicity of the triclosan. However, due to the lack of commercial triclosan phase II metabolite standards, many studies have evaluated the total amount of triclosan by enzymatic hydrolysis of triclosan binding products, which is an internationally common expedient, but have failed to accurately reveal the process of migratory transformation of triclosan in organisms.
At present, only two international reports about a synthesis method of triclosan sulfonic acid exist, and the prepared product can only be used for qualitative determination of triclosan sulfonic acid in a sample and cannot meet the requirement of accurate quantification. Wherein, the preparation process of the biosynthesis method is complex and the yield is low; the chemical synthesis method has the advantages that the toxicity of a reaction solvent used in the chemical synthesis method is high, the requirement on the reaction temperature is high, a semi-preparative liquid chromatograph is used in the separation and purification process, the separation time is long, the efficiency is low, and most importantly, excessive ammonium acetate is introduced into a semi-preparative liquid chromatograph mobile phase, so that the purity of the triclosan sulfonic acid cannot meet the quantitative requirement. In addition, triclosan sulfonic acid cannot exist stably in a solid state, which aggravates the difficulty of accurately quantifying triclosan sulfonic acid in a sample as a standard, and thus, the preparation of solid triclosan sulfonate is an important way to solve the problem. However, excessive sodium ions and potassium ions are easily introduced in the synthesis process of salts such as sodium triclosan sulfonate, potassium triclosan sulfonate and the like and cannot be completely removed.
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 ammonium triclosan sulfonate and a preparation method and application thereof, and aims to solve the problems that the existing standard triclosan sulfonic acid for detecting triclosan sulfonic acid cannot exist stably in a solid state, and sodium triclosan sulfonate or potassium triclosan sulfonate cannot be completely removed due to excessive sodium ions or potassium ions which are easily introduced in the synthesis process, so that the purity is not sufficient, and the accurate quantification of the triclosan sulfonic acid in a sample cannot be carried out.
The technical scheme of the invention is as follows:
ammonium triclosan sulfonate, wherein the structural formula of the ammonium triclosan sulfonate is shown in the specification
Figure GDA0002716984790000021
A method for preparing ammonium triclosan sulfonate as described above, comprising:
step A, adding triclosan and sulfur trioxide pyridine complex with the mass ratio of 1:2-4 into a solvent, and stirring to react at the temperature of 15-40 ℃ to obtain triclosan sulfonic acid reaction liquid;
step B, carrying out reduced pressure concentration on the triclosan sulfonic acid reaction solution, and carrying out purification treatment to obtain a purified triclosan sulfonic acid solution;
and C, reacting the purified triclosan sulfonic acid solution with excessive ammonia water, concentrating, and freeze-drying to obtain the triclosan ammonium sulfonate.
The preparation method of the triclosan ammonium sulfonate comprises the step A, wherein the solvent is N, N-dimethylformamide.
In the preparation method of ammonium triclosan sulfonate, in the step B, the purification treatment process specifically comprises the following steps:
step B1, activating the solid phase extraction column by using methanol and water in sequence, and then adding the concentrated triclosan sulfonic acid reaction solution into the activated solid phase extraction column;
step B2, leaching the solid phase extraction column by using methanol-water mixed solvents with the volume ratio of 1:9, 3:7 and 1:1 in sequence, eluting the solid phase extraction column liquid by using the methanol-water mixed solvent with the volume ratio of 7:3, and finally cleaning the solid phase extraction column by using methanol;
and step B3, decompressing and concentrating the methanol-water mixed eluent with the volume ratio of 7:3, adding the concentrated methanol-water mixed eluent with the volume ratio of 7:3 into the activated solid phase extraction column, repeating the step B2 for further purification, and obtaining the 7:3 methanol-water mixed eluent which is the purified triclosan sulfonic acid solution after further purification.
The preparation method of the ammonium triclosan sulfonate comprises the following specific steps of: and (3) reacting the purified triclosan sulfonic acid solution with excessive ammonia water to obtain an ammonium triclosan sulfonate reaction solution, concentrating under reduced pressure, and freeze-drying the concentrated ammonium triclosan sulfonate reaction solution to obtain a solid compound ammonium triclosan sulfonate.
In the preparation method of the ammonium triclosan sulfonate, in the step B1, the specification of the solid phase extraction column is Generik H2P solid phase extraction column, 75mL and 10 g.
The method for detecting the triclosan sulfonic acid in the biological sample adopts the ammonium triclosan sulfonate as a standard substance and adopts an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer to detect the triclosan sulfonic acid in the biological sample.
The method for detecting the triclosan sulfonic acid in the biological sample, wherein the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprises an ultra-high performance liquid chromatography system and a mass spectrometry system;
the parameters of the ultra-high performance liquid chromatography system comprise: an ACQUITY UPLC BEH C18 chromatographic column with a specification of 1.7 μm, 100mm × 2.1mm, Waters; the mobile phase comprises acetonitrile and 2mM ammonium acetate water solution with flow rate of 0.30mL min–1(ii) a The column temperature was 35 ℃; the sample injection amount is 10 mu L;
the parameters of the mass spectrometry system include: the ionization mode is an electrospray negative ionization mode, the data acquisition mode is a multi-reaction monitoring mode, the spray voltage is 2600V, the temperature of an ion transmission tube is 350 ℃, and the temperature of a sprayer is 300 ℃.
The method for detecting the triclosan sulfonic acid in the biological sample comprises the steps of taking 3.61min as the chromatographic retention time of the triclosan sulfonic acid, and taking m/z368.89 → 288.89 and m/z366.89 → 286.89 as the monitoring ion pair of the triclosan sulfonic acid, and qualitatively detecting the triclosan sulfonic acid in the biological sample.
The method for detecting the triclosan sulfonic acid in the biological sample comprises the steps of taking m/z366.89 → 286.89 as a monitoring ion pair for quantifying the triclosan sulfonic acid, quantitatively detecting the triclosan sulfonic acid in the biological sample by using the peak area of a chromatographic peak, and calculating by using a standard curve method to obtain a quantitative detection result.
Has the advantages that: the ammonium triclosan sulfonate can stably exist in a solid state, replaces triclosan sulfonic acid as a standard substance for detecting the triclosan sulfonic acid in a biological sample, can meet the requirement of accurate quantitative detection, has high detection sensitivity, and lays a foundation for researching the migration and transformation of triclosan in a living body and the mechanism causing biological toxicity. The preparation method of the triclosan ammonium sulfonate has the advantages of short synthetic route, simple reaction device, convenience and rapidness in operation and the like, and the obtained ammonium salt has strong stability and high purity, and the product is separated and purified by adopting a solid-phase extraction method.
Drawings
FIG. 1 shows a chromatogram-mass spectrum (a) of a triclosan sulfonic acid reaction solution obtained in step (1) of example 1 in the Full MS mode and a chromatogram-mass spectrum (b) in the PRM mode.
FIG. 2 is a chromatogram obtained by measuring the eluents, eluents and washing solutions in Full MS mode after the first solid-phase extraction separation in step (2) of example 1 of the present invention.
FIG. 3 is a chromatogram obtained from the measurement of the eluent and the washing solution in the Full MS mode after the second solid-phase extraction and purification in step (2) of example 1 of the present invention.
FIG. 4 is a NMR chart of a final product obtained in step (3) of example 1 of the present invention.
FIG. 5 is a chromatogram-mass spectrum of the final product obtained in step (3) of example 1 of the present invention, measured in Full MS mode.
FIG. 6 is a chromatogram (a) of a biological sample of a control group measured in MRM mode and a chromatogram (b) of a biological sample of an exposure group measured in MRM mode in example 2 of the present invention.
Detailed Description
The invention provides ammonium triclosan sulfonate and a preparation method and application thereof, 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.
Ammonium triclosan sulfonate, wherein the structural formula of the ammonium triclosan sulfonate is shown in the specification
Figure GDA0002716984790000051
The ammonium triclosan sulfonate can stably exist in a solid state, replaces triclosan sulfonic acid as a standard substance for detecting the triclosan sulfonic acid in a biological sample, can meet the requirement of accurate quantitative detection, has high detection sensitivity, and lays a foundation for researching the migration and transformation of triclosan in a living body and the mechanism causing biological toxicity.
A preferred embodiment of the method for preparing ammonium triclosan sulfonate, comprises:
step A, adding triclosan and sulfur trioxide pyridine complex with the mass ratio of 1:2-4 (preferably 1:2-3) into a solvent (preferably N, N-dimethylformamide), and stirring to react at the temperature of 15-40 ℃ (preferably 25-35 ℃) to obtain triclosan sulfonic acid reaction liquid;
step B, concentrating the triclosan sulfonic acid reaction solution to 1-5mL (preferably 2-4mL) under reduced pressure, and purifying to obtain a purified triclosan sulfonic acid solution;
and C, reacting the purified triclosan sulfonic acid solution with excessive ammonia water, concentrating, and freeze-drying to obtain the triclosan ammonium sulfonate.
In the preparation method of ammonium triclosan sulfonate, in the step B, the purification treatment process specifically comprises the following steps:
step B1, activating the solid phase extraction column by using methanol and water in sequence, and then adding the concentrated triclosan sulfonic acid reaction solution into the activated solid phase extraction column; preferably, the specification of the solid phase extraction column is Generik H2P solid phase extraction column, 75mL, 10 g;
step B2, leaching the solid phase extraction column by using methanol-water mixed solvents with the volume ratio of 1:9, 3:7 and 1:1 in sequence, eluting the solid phase extraction column by using the methanol-water mixed solvents with the volume ratio of 7:3, and finally cleaning the solid phase extraction column by using methanol;
and step B3, concentrating the methanol-water mixed eluent with the volume ratio of 7:3 to 2-6mL (preferably 3-5mL) at 45 ℃ under reduced pressure, adding the concentrated methanol-water mixed eluent with the volume ratio of 7:3 into an activated solid phase extraction column, repeating the step B2 for further purification, and obtaining the 7:3 methanol-water mixed eluent which is the purified triclosan sulfonic acid solution after further purification.
The step C specifically comprises the following steps: and reacting the purified triclosan sulfonic acid solution with excessive ammonia water to obtain an ammonium triclosan sulfonate reaction solution, concentrating the solution to 1-4mL under reduced pressure, and freeze-drying the concentrated ammonium triclosan sulfonate reaction solution to obtain a solid compound ammonium triclosan sulfonate.
The preparation method of the triclosan ammonium sulfonate has the advantages of short synthetic route, simple reaction device, convenience and rapidness in operation and the like, and the obtained ammonium salt has strong stability and high purity, and the product is separated and purified by adopting a solid-phase extraction method.
In the preferred embodiment of the method for detecting triclosan sulfonic acid in a biological sample, ammonium triclosan sulfonate is used as a standard substance, and an ultra performance liquid chromatography-triple quadrupole tandem mass spectrometer is used for detecting triclosan sulfonic acid in the biological sample.
Specifically, the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer comprises an ultra-high performance liquid chromatography system and a mass spectrometry system;
the parameters of the ultra-high performance liquid chromatography system comprise: an ACQUITY UPLC BEH C18 chromatographic column with a specification of 1.7 μm, 100mm × 2.1mm, Waters; the mobile phase comprises acetonitrile and 2mM ammonium acetate water solution with flow rate of 0.30mL min–1(ii) a The column temperature was 35 ℃; the sample injection amount is 10 mu L;
the parameters of the mass spectrometry system include: the ionization mode is electrospray negative ionization (ESI)-) The data acquisition mode is a multi-reaction monitoring mode (MRM), the spraying voltage is 2600V, the temperature of an ion transmission tube is 350 ℃, and the temperature of a sprayer is 300 ℃.
Specifically, the chromatographic retention time of the triclosan sulfonic acid is 3.61min, and the monitoring ion pair of the triclosan sulfonic acid is m/z368.89 → 288.89 and m/z366.89 → 286.89, so that the triclosan sulfonic acid in the biological sample can be qualitatively detected.
Specifically, m/z366.89 → 286.89 is taken as a monitoring ion pair for quantifying the triclosan sulfonic acid, the peak area of a chromatographic peak is taken as the quantitative detection of the triclosan sulfonic acid in a biological sample, and the quantitative detection result is obtained by calculation with a standard curve method; the chromatographic peak refers to the chromatographic peak corresponding to the chromatographic retention time of the triclosan sulfonic acid.
The method for detecting the triclosan sulfonic acid in the biological sample by taking the ammonium triclosan sulfonate as the standard substance can meet the requirement of accurate quantitative detection, has high detection sensitivity, and lays a foundation for researching the migration and transformation of triclosan in a biological body and the mechanism causing biological toxicity.
The present invention will be described in detail below with reference to examples.
EXAMPLE 1 preparation of ammonium triclosan sulfonate
(1) The preparation of the triclosan sulfonic acid reaction solution, the reaction formula for synthesizing the triclosan sulfonic acid is
Figure GDA0002716984790000071
50mg of triclosan and 100mg of sulfur trioxide pyridine complex are put into a single-mouth bottle, 2mL of N, N-Dimethylformamide (DMF) is added, and the mixture is magnetically stirred at the temperature of 25 ℃ for reaction for 24 hours to obtain a triclosan sulfonic acid reaction solution.
A small amount of triclosan sulfonic acid reaction solution is diluted to 1/200 of the original concentration with methanol, and after ultra-high speed centrifugation (15,000rpm, 10min), the triclosan sulfonic acid reaction solution is identified by an ultra-performance liquid chromatography (UPLC) -Q extraction combined quadrupole/orbital trap 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: 0.30 mL/min–1The sample injection amount is as follows: 10 μ L. The mobile phase A is acetonitrile, and the mobile phase B is an aqueous phase containing 2mM ammonium acetate; elution gradient started with 20% A + 80% B (v/v) as mobile phase for 1.0 min; 80% A + 20% B (v/v) at 2.0min as mobile phase and held for 6.0 min; at 6.5min 100% A + 0% B (v/v) as mobile phase and hold to 7.5 min; the solution returns to the initial state at 8.0min, and 20% A + 80% B (v/v) is used as a mobile phase and lasts for 2.0 min. QE mass spectrometry system using negative ionization mode, and Full scan mode (Full MS) and parallel reaction monitoring mode (PRM) (to35Cl as an example), the structure of each component in the triclosan sulfonic acid reaction solution is identified, the chromatogram-mass spectrum of each component measured under Full MS is shown in figure 1(a), and the chromatogram-mass spectrum of each component measured under PRM mode is shown in figure 1 (b). The results showed that the triclosan sulfonic acid reaction solution contained the triclosan sulfonic acid formed, and the chromatographic retention time of the triclosan sulfonic acid was 3.61 min.
(2) Preparation of purified triclosan sulfonic acid solution: (a) concentrating the triclosan sulfonic acid reaction solution in the step 1 to 3mL under reduced pressure, sequentially activating a Generic H2P solid phase extraction column by using 100mL of methanol and 100mL of water, and adding the concentrated triclosan sulfonic acid reaction solution into the activated Generic H2P solid phase extraction column; (b) leaching the Generick H2P solid-phase extraction column with 100mL of 10%, 30% and 50% methanol/water (v/v), eluting the target compound in the Generick H2P solid-phase extraction column with 100mL of 70% methanol/water (v/v), and finally washing the Generick H2P solid-phase extraction column with 100mL of 100% methanol; (c) the 70% methanol/water (v/v) eluate was concentrated to about 4-5 mL by rotary evaporation at 45 ℃ under reduced pressure, and the step (b) was repeated to further purify the concentrated 70% methanol/water (v/v) eluate using a Generic H2P solid phase extraction column to obtain a purified triclosan sulfonic acid solution.
Taking each eluent, eluent and cleaning solution in the separation process in the step (b), centrifuging at ultra high speed (15,000rpm for 10min), and analyzing by using UPLC-QE mass spectrum in Full MS mode according to the method, wherein chromatograms of the eluent, the eluent and the cleaning solution in the separation process in the Full MS mode are shown in FIG. 2. The results show that the triclosan sulfonic acid is mainly present in the 70% methanol/water eluent, and contains main impurities which are raw material triclosan; according to the peak area, the purity of the triclosan sulfonic acid is calculated to be 96.2%. After centrifuging at ultra high speed (15,000rpm, 10min) the 70% methanol/water (v/v) eluate and 100% methanol wash from (c) separation, the above analysis was performed by UPLC-QE mass spectrometry in Full MS mode, and the chromatogram obtained from the 70% methanol/water (v/v) eluate and 100% methanol wash in Full MS mode during separation is shown in fig. 3. The results show that the purity of the triclosan sulfonic acid in the 70% methanol/water (v/v) eluent is more than 99%, and the further purified 70% methanol/water (v/v) eluent is the purified triclosan sulfonic acid solution.
It should be noted that triclosan sulfonic acid cannot exist stably in solid form, and if the eluate is directly dried, the triclosan sulfonic acid will be completely converted into triclosan as a raw material.
(3) The preparation of the ammonium triclosan sulfonate and the reaction formula for synthesizing the ammonium triclosan sulfonate are
Figure GDA0002716984790000091
Adding 8mL of ammonia water into the purified triclosan sulfonic acid solution, fully mixing uniformly, performing rotary evaporation at 45 ℃ under reduced pressure to concentrate to 1mL, performing freeze drying at-50 ℃ under reduced pressure, and completely removing excessive ammonia water in a gaseous form during the freeze drying under reduced pressure to obtain a final product which is a solid.
With methane-d4Determining the NMR spectrum of the final product as a solvent, wherein the NMR spectrum of the final product is shown in FIG. 4; 1H-NMR (methane-d)4400MHz) δ: 7.70(d,1H), 7.51(d,1H), 7.22(dd,1H), 7.12(dd,1H), 6.87(t, 2H). The final product was dissolved in methanol and mass spectrometrically detected in Full MS mode, and the chromatogram-mass spectrum of the final product in Full MS mode is shown in fig. 5, which corresponds to the theoretical value. Indicating that the triclosan sulfonic acid is completely converted to the ammonium salt and is stable.
EXAMPLE 2 detection of triclosan sulfonic acid in biological samples
(1) Optimizing mass spectrometry parameters associated with triclosan sulfonic acid
A UPLC-TSQ triple quadrupole tandem mass spectrometer (Thermo Fisher Scientific, USA) is used to establish a triclosan sulfonic acid detection method. The conditions of the UPLC ultra high performance liquid chromatography system were as described in example 1; TSQ mass spectrometry system using electrospray negative ionization mode (ESI)-) The multiple reaction monitoring mode (MRM) scan was selected, the spray voltage was 2600V, the ion transfer tube temperature was 350 deg.C, and the sprayer temperature was 300 deg.C. The ammonium triclosan sulfonate prepared above is optimized to obtain triclosan sulfonic acid with the ion pairs m/z366.89 → 286.89 (16 eV for collision energy) and m/z368.89 → 288.89 (16 eV for collision energy), and can be used as a qualitative monitoring ion pair. Of these, m/z366.89 → 286.89 was selected as a monitoring ion pair for quantification.
(2) Preparing a biological sample
Taking human normal liver cells L02 in logarithmic growth phase, adding cell culture solution to adjust triclosan exposure dose to 1 mu mol. L–1. After 48h exposure the cell culture medium was removed, the cells were washed 2 times with Phosphate Buffered Saline (PBS), trypsinized and centrifuged (1000rpm, 5min), after matrix removal, pre-cooled 80% methanol/water (containing isotope internal standard TCS-d) was added3) Liquid nitrogen counterFreezing and thawing the cells for 5 times, centrifuging (15,000rpm, 10min, 4 ℃) to obtain a cell extracting solution to be detected, and taking the cell extracting solution to be detected as a biological sample of the exposure group; meanwhile, a cell extract without exposing triclosan is set as a biological sample of a control group.
(3) Detection of triclosan sulfonic acid in biological samples
The ammonium triclosan sulfonate prepared by the invention is used as a standard substance, the detection conditions are the same as those in the step (1) of the embodiment, and 3.61min is taken as the chromatographic retention time of triclosan sulfonic acid; m/z366.89 → 286.89 and m/z368.89 → 288.89 are used as qualitative monitoring ion pairs of triclosan sulfonic acid; the quantitative monitoring ion pair of m/z366.89 → 286.89 triclosan sulfonic acid; and (3) respectively detecting the biological samples of the exposure group and the control group by using an UPLC-TSQ triple quadrupole tandem mass spectrometer, wherein the chromatogram obtained by measuring the biological sample of the control group in the MRM mode is shown in fig. 6(a), and the chromatogram obtained by measuring the biological sample of the exposure group in the MRM mode is shown in fig. 6 (b). The detection result shows that the triclosan sulfonic acid is not detected in the biological sample of the control group; the generation of triclosan sulfonic acid was detected in the biological samples in the exposed group, the content of triclosan sulfonic acid in the biological samples in the exposed group was quantified as the peak area of the chromatographic peak corresponding to a chromatographic retention time of 3.61min, and the concentration of triclosan sulfonic acid was calculated to be 0.174. mu. mol. L by standard curve method–1(ii) a The original concentration of triclosan is 2.08 mu mol.L–1Then the molar percentage of triclosan sulfonic acid in the biological sample is 8.4%, indicating that the cell is undergoing a sulfonated metabolic reaction with triclosan.
In conclusion, the ammonium triclosan sulfonate provided by the invention can stably exist in a solid state, replaces triclosan sulfonic acid as a standard substance for detecting triclosan sulfonic acid in a biological sample, can meet the requirement of accurate quantitative detection, has high detection sensitivity, and lays a foundation for researching the migration and transformation of triclosan in a living body and the mechanism of causing biotoxicity. The preparation method of the triclosan ammonium sulfonate has the advantages of short synthetic route, simple reaction device, convenience and rapidness in operation and the like, and the obtained ammonium salt has strong stability and high purity, and the product is separated and purified by adopting a solid-phase extraction method.
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 (3)

1. A preparation method of ammonium triclosan sulfonate is characterized by comprising the following steps:
step A, adding triclosan and sulfur trioxide pyridine complex with the mass ratio of 1:2-4 into a solvent, and stirring to react at the temperature of 15-40 ℃ to obtain triclosan sulfonic acid reaction liquid;
step B, carrying out reduced pressure concentration on the triclosan sulfonic acid reaction solution, and carrying out purification treatment to obtain a purified triclosan sulfonic acid solution;
step C, reacting the purified triclosan sulfonic acid solution with excessive ammonia water, concentrating, and freeze-drying to obtain triclosan ammonium sulfonate;
in the step B, the purification process specifically includes:
step B1, activating the solid phase extraction column by using methanol and water in sequence, and then adding the concentrated triclosan sulfonic acid reaction solution into the activated solid phase extraction column;
step B2, leaching the solid phase extraction column by using methanol-water mixed solvents with the volume ratio of 1:9, 3:7 and 1:1 in sequence, eluting the solid phase extraction column by using the methanol-water mixed solvents with the volume ratio of 7:3, and finally cleaning the solid phase extraction column by using methanol;
step B3, decompressing and concentrating the methanol-water mixed eluent with the volume ratio of 7:3, adding the concentrated methanol-water mixed eluent with the volume ratio of 7:3 into an activated solid phase extraction column, repeating the step B2 for further purification, and obtaining the 7:3 methanol-water mixed eluent which is the purified triclosan sulfonic acid solution after further purification;
in step B1, the specification for the solid phase extraction column was Generik H2P solid phase extraction column, 75mL, 10 g.
2. The method of claim 1, wherein in step a, the solvent is N, N-dimethylformamide.
3. The method for preparing ammonium triclosan sulfonate according to claim 1, wherein step C specifically comprises: and (3) reacting the purified triclosan sulfonic acid solution with excessive ammonia water to obtain an ammonium triclosan sulfonate reaction solution, concentrating under reduced pressure, and freeze-drying the concentrated ammonium triclosan sulfonate reaction solution to obtain a solid compound ammonium triclosan sulfonate.
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