A method for detecting stable isotopes of carbon and nitrogen in sulfamethoxazole monomers

Abstract

This invention discloses a method for detecting the stable carbon and nitrogen isotopes of sulfamethoxazole monomers, belonging to the field of analytical chemistry. By optimizing the derivatization conditions of sulfamethoxazole, this invention achieves gas chromatography separation and detection, and establishes an analytical method for the stable carbon and nitrogen isotopes of sulfamethoxazole monomers using GC-C-IRMS. This fills a gap in the field of antibiotic stable isotope analysis and lays the foundation for in-depth research on the migration and transformation processes of antibiotics in the environment.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, and more specifically, relates to a method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer. Background Technology

[0002] Sulfamethoxazole (SMX), also known as sulfamethoxazole, is a commonly used broad-spectrum sulfonamide antibiotic. It primarily inhibits bacterial growth and reproduction by suppressing the synthesis of bacterial membrane proteins, and is widely used to treat bacterial infections of the respiratory, urinary, and digestive tracts. However, with the widespread use of SMX, its environmental residues have become a major concern. After entering the bodies of animals and humans, SMX is not completely absorbed; 30%-90% of SMX is excreted in feces and urine as the parent drug or metabolites. Wastewater from sewage treatment plants and animal manure composting are the main pathways for SMX to enter the environment. In the environment, SMX readily undergoes migration and transformation behaviors such as water degradation, photodegradation, microbial degradation, adsorption and desorption, surface runoff transport, leaching, and plant uptake. Therefore, a thorough understanding of the complex environmental sources and fate of SMX is crucial for studying its pollution characteristics, exposure risks, and mitigation methods.

[0003] Traditional chemical methods elucidate the migration and transformation processes of organic pollutants and metabolites through qualitative and quantitative studies. However, deducing degradation pathways from metabolites is uncertain. In complex field environments, dilution, volatilization, or adsorption can reduce the concentration of organic pollutants, creating a false impression of degradation. Compound-specific isotope analysis (CSIA) is an analytical technique that studies changes in the ratios of naturally occurring stable isotopes (such as carbon, oxygen, nitrogen, and sulfur) caused by isotopic fractionation during compound migration and transformation, revealing the sources, transformation patterns, and degradation mechanisms of organic matter. This method effectively overcomes the limitations of traditional methods and has been applied to the study of organic pollutants in the environment. For example, Chinese invention patent application No. 201610542036.1, filed on July 11, 2016, discloses a method for analyzing the stable carbon isotopes of polybrominated diphenyl ethers in fish; Chinese invention patent application No. 202010098354.X, filed on February 18, 2020, discloses a method for separating and purifying sixteen polycyclic aromatic hydrocarbons in soil and a method for detecting their stable carbon isotopes; Chinese invention patent application No. 202110137824.3, filed on February 1, 2021, discloses a method for determining the stable carbon isotopes of bromophenols and its application; and Chinese invention patent application No. 202210820078.2, filed on July 13, 2022, discloses a method for detecting the stable carbon isotope composition of low-trace concentration benzene series compounds in water.

[0004] Gas chromatography-combustion (GC)-infrared ionization mass spectrometry (CSIA) is a commonly used instrument in laboratories for detecting stable isotope ratios of organic compounds. First, gas chromatography (GC) separates the organic compounds from the sample. Then, a combustion furnace (C) further converts the elements in the organic compounds into gases that meet analytical requirements. The analyte gases are ionized and detected by isotope ratio mass spectrometry (IRMS), which outputs the final stable isotope ratio. CSIA based on GC-C-IRMS analysis is mainly suitable for compounds with simple molecular structures, low boiling points, high volatility, and low decomposition at high temperatures, such as volatile organic compounds, chlorinated hydrocarbons, pesticides, and petroleum pollutants. However, antibiotics have complex and diverse chemical structures and are generally not separable by gas chromatography. To date, there are very few reports on the detection of stable isotope ratios for antibiotics. For example, SMX is a nitrogen-containing heterocyclic compound with high polarity and hydrogen bonding, making it unstable under gas-phase conditions and prone to fragmentation and rearrangement. In addition, SMX molecules have a relatively large molecular mass, requiring high temperatures for evaporation and separation in gas chromatography. This can lead to further decomposition and degradation of the molecules, making them difficult to separate and detect. Summary of the Invention

[0005] 1. The problem to be solved

[0006] To address the limitations of existing gas chromatography methods in separating and detecting antibiotics in the environment and in studying antibiotic migration and transformation processes, this invention provides a method for detecting the stable carbon and nitrogen isotopes of sulfamethoxazole monomers. This invention achieves gas chromatographic separation and detection by derivatizing sulfamethoxazole and establishes a GC-C-IRMS method for analyzing the stable carbon and nitrogen isotopes of sulfamethoxazole monomers. This fills a gap in the field of antibiotic stable isotope analysis and lays the foundation for in-depth research on the migration and transformation processes of antibiotics in the environment.

[0007] 2. Technical Solution

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer, comprising the following steps:

[0010] S10. Sulfamethoxazole is dissolved in the first solvent to prepare a solution of sulfamethoxazole in the first solvent. Trimethylsilyldiazomethane (TMSD) is used as a derivatizing agent to derivatize the solution of sulfamethoxazole in the first solvent to obtain a derivatized solution of sulfamethoxazole in the first solvent.

[0011] S20. Remove the first solvent and redissolve the derivatized sulfamethoxazole in the second solvent to obtain a solution of derivatized sulfamethoxazole in the second solvent.

[0012] S30. The derivatized sulfamethoxazole solution in the second solvent is separated by gas chromatography, and then burned in a combustion furnace to convert it into CO2 and N2. The carbon stable isotope ratio and nitrogen stable isotope ratio are determined by IRMS.

[0013] Preferably, the first solvent is methanol; the second solvent is one or more of acetone, n-hexane, and methanol.

[0014] Preferably, in step S10, the molar ratio of trimethylsilyl diazomethane to sulfamethoxazole is (9-95):1.

[0015] More preferably, the molar ratio of the trimethylsilyl diazomethane to sulfamethoxazole is (57-95):1.

[0016] Preferably, in step S10, the reaction time of the derivatization reaction is 15 to 120 minutes.

[0017] More preferably, the reaction time of the derivatization reaction is 15 minutes.

[0018] Preferably, in step S10, the reaction temperature of the derivatization reaction is 20℃~80℃.

[0019] More preferably, the reaction temperature of the derivatization reaction is 40°C.

[0020] Preferably, in step S10, the concentration of the sulfamethoxazole solution in the first solvent is 200–2000 mg / L.

[0021] Preferably, the specific process of step S30 is as follows: the derivatized sulfamethoxazole solution in the second solvent is injected twice into the gas chromatograph for separation, and then enters the combustion furnace to be converted into CO2 and N2. The carbon stable isotope ratio and nitrogen stable isotope ratio are determined by IRMS, wherein before determining the nitrogen stable isotope ratio by IRMS, the CO2 converted by combustion in the combustion furnace is removed by liquid nitrogen and hydrazine device.

[0022] Preferably, in step S30, the injection port temperature of the gas chromatograph is 250°C, the carrier gas is helium with a flow rate of 1 mL / min, the injection volume is 4 μL, and the injection is splitless. The temperature program of the gas chromatograph is as follows: initial temperature 80°C, hold for 2 min; increase to 180°C at 30°C / min, hold for 2 min; increase to 280°C at 10°C / min, hold for 5 min; and the preparation time is 10 min.

[0023] Preferably, in step S30, the temperature in the combustion furnace is 1000℃~1100℃.

[0024] Preferably, in step S30, the method detection limit for determining the carbon stable isotope ratio by IRMS is 150 mg / L; and the method detection limit for determining the nitrogen stable isotope ratio by IRMS is 400 mg / L.

[0025] 3. Beneficial effects

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The present invention provides a method for detecting carbon and nitrogen stable isotopes of sulfamethoxazole monomers. It establishes for the first time the analysis and determination of carbon and nitrogen stable isotopes of antibiotic monomers, filling the gap that GC-C-IRMS cannot detect the stable isotope ratio characteristics of high-boiling-point organic compounds. At the same time, it effectively avoids the limitations of single-element isotope detection, ensuring a more comprehensive revelation of the source, transformation law and degradation mechanism of organic compounds.

[0028] (2) The present invention provides a method for detecting carbon and nitrogen stable isotopes of sulfamethoxazole monomers, which completes the derivatization of antibiotic SMX in one step by selecting appropriate derivatization conditions, and realizes qualitative and quantitative analysis of derivatized SMX by GC-MS.

[0029] (3) The present invention provides a method for detecting carbon and nitrogen stable isotopes of sulfamethoxazole monomer. The method is simple to operate, efficient, and reproducible. It can quickly and reliably determine the carbon and nitrogen stable isotope characteristics of SMX in environmental samples, laying the foundation for studying the migration and transformation process of sulfonamide antibiotics. Attached Figure Description

[0030] Figure 1 (a)-1(b) are gas chromatograms of derivatized SMX solution (a) and control underivatized SMX solution (b);

[0031] Figure 2 (a)-2(b) are full scan mass spectra of derivatized SMX solution (a) and SMX standard (b);

[0032] Figure 3 (a)-3(b) are the carbon (a) and nitrogen (b) stable isotope spectra of the derivatized SMX monomers determined by GC-C-IRMS;

[0033] Figure 4 (a)-4(b) represent the δ values ​​at different SMX concentrations. 13 C value (a) and δ 15 N value (b) and amplitude value;

[0034] Figure 5(a)-5(b) are δ under multiple injection conditions 13 C value (a) and δ 15 N value (b) and δ detected by elemental analysis isotope ratio mass spectrometry (EA-IRMS) 13 C value (a) and δ 15 N value (b);

[0035] Figure 6 δ under different TMSD excess factors 13 C value and δ 15 N value;

[0036] Figure 7 (a)-7(b) are carbon stable isotope spectra of derivatized SMX monomers determined by GC-C-IRMS when the excess ratios are 9(a) and 19(b);

[0037] Figure 8 (a)-8(b) represent the δ values ​​at different derivatization reaction temperatures (a) and reaction times (b). 13 C value. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] Instruments and reagents: Dry bath nitrogen purging apparatus, Hangzhou Ruicheng Instrument Co., Ltd. ND200-2; Oven, Shanghai Yiheng Scientific Instrument Co., Ltd.; Vortex mixer, Raytheon Technologies, USA; Gas chromatography-mass spectrometry detector coupled with instrument, Agilent 7890A / 5975C, USA; Gas chromatography interface combustion furnace tandem isotope ratio mass spectrometer, Thermo Fisher Scientific, 253Plus, Germany; Methanol (chromatographic grade) and acetone (chromatographic grade) were purchased from Merck, USA; SMX standard was purchased from Dr. Ehrenstorfer, Germany, with a purity ≥99%; TMSD reagent was purchased from Aladdin, USA, with a concentration of 1.5M n-hexane.

[0040] Example 1

[0041] This embodiment mainly examines the qualitative and quantitative analysis of derivatized SMX by GC-MS.

[0042] (1) Weigh 20 mg of sulfamethoxazole (SMX) standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol as the primary solvent to prepare a 2000 mg / L SMX stock solution. Dilute it to 1 mL to obtain a 50–800 mg / L SMX methanol solution;

[0043] (2) Add the derivatization reagent trimethylsilyl diazomethane (TMSD) to the SMX methanol solution. The molar ratio of TMSD to SMX is 76:1. The derivatization reaction is carried out in a 60°C oven in the dark for 120 min.

[0044] (3) After the derivatization reaction was completed, the methanol solvent was dried in a steady nitrogen flow. The methanol solvent was reconstituted with 1 mL of acetone as the second solvent and vortexed for 1 min to completely dissolve it. The solution was then passed through a 0.22 μm organic filter membrane and transferred to a 2 mL brown sample vial to obtain the derivatized SMX acetone solution, which was stored at -20 °C for further analysis.

[0045] (4) Qualitative analysis of the derivatized SMX acetone solution was performed using GC-MS, where:

[0046] Gas chromatography parameters: column: HP-5MS (30m x 250μm x 0.25μm), injection port temperature: 250℃, carrier gas: helium, flow rate: 1mL / min, injection volume: 1μL, splitless injection; gas chromatography temperature program: initial temperature 80℃, hold for 2 min; increase to 180℃ at 10℃ / min, hold for 2 min; increase to 280℃ at 10℃ / min, hold for 5 min; run at 310℃ for 2 min.

[0047] Mass spectrometry detector monitoring method: electron impact ion source (EI source), electron energy value 70EV, mass spectrometry ion source temperature value 300℃, quadrupole temperature value 180℃; full scan mode analysis mass-to-charge ratio range 30~500, selected ion scan mode (SIM) selects two characteristic ions with high abundance.

[0048] like Figure 1 As shown in (a), the derivatized SMX acetone solution can be effectively separated and detected by GC-MS with a retention time of 21.06 min. Under the current GC-MS detection conditions, the target compound exhibits sensitive signal response, good peak shape, and minimal impurity interference; while... Figure 1 In (b), the control underrivatized SMX solution did not elute under the same conditions.

[0049] Full scan mass spectra of derivatized SMX and SMX standard are shown below. Figure 2As shown in (a)-2(b), the derivatized SMX possesses the characteristic ions of sulfonamide antibiotics (m / z 65, m / z 92, m / z 108, m / z 156, m / z 162), indicating that the target compound is a derivative of SMX. Since there is no commercially available standard for this derivatized SMX, correlation analysis was performed between the concentration of the SMX standard and the peak area of ​​the derivatized SMX in SIM mode. The results showed that the derivatized SMX exhibited a good linear relationship within the concentration range (50-800 mg / L), with a correlation coefficient greater than 0.999, indicating that the derivatization method of this embodiment can quantitatively convert SMX without revealing any obvious side reaction pathways.

[0050] Example 2

[0051] This embodiment of a method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer includes the following steps:

[0052] S10. Weigh 20 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol to prepare a 2000 mg / L SMX stock solution. Dilute the stock solution to 1 mL to prepare SMX methanol solutions with concentrations of 200 mg / L and 1400 mg / L. Add the derivatization reagent TMSD to the SMX methanol solution. The molar ratio of TMSD to SMX is 76:1. Incubate the derivatization reaction in a 60 °C oven in the dark for 120 min.

[0053] After the derivatization reaction was completed, the methanol solvent was dried under a steady nitrogen flow. It was then reconstituted with 1 mL of acetone and vortexed for 1 min to completely dissolve it. The solution was then passed through a 0.22 μm organic filter membrane and transferred to a 2 mL brown sample vial to obtain the derivatized SMX acetone solution, which was stored at -20 °C for further analysis.

[0054] S30. The stable isotopes of monomeric carbon and nitrogen in the derivatized SMX solution were analyzed and determined using a gas chromatography-interface combustion furnace isotope ratio mass spectrometer (GC-C-IRMS).

[0055] The derivatized SMX acetone solution was introduced into a GC for separation using an autosampler. The GC inlet temperature was 250°C, the carrier gas was helium at a flow rate of 1 mL / min, and the GC injection volume was 4 μL. The gas chromatography temperature program was as follows: initial temperature 80°C, hold for 2 min; increase to 180°C at 30°C / min, hold for 2 min; increase to 280°C at 10°C / min, hold for 5 min; preparation run time was 10 min; and then the sample was introduced into a combustion furnace, where it was converted into CO2 and N2 at a temperature of 1020°C.

[0056] The carbon stable isotope ratios and nitrogen stable isotope ratios were determined by IRMS. The carbon stable isotope ratio δ0.05 of the sample was determined.13 The calculation process for C(‰) is as follows: δ 13 C(‰)=((R sample / R standard )-1)×1000, R sample and R standard These are the sample and the reference standard sample, Vienna Pee Dee Belemnite (VDPB). 13 C / 12 C value.

[0057] Nitrogen temperature isotope ratio δ of the sample 15 The calculation process for N(‰) is as follows: δ 15 N(‰)=((R sample / R standard )-1)×1000, R sample and R standard These are the nitrogen (Air-N2) samples and reference standard samples, respectively. 15 N / 14 When determining nitrogen isotopes, an additional liquid nitrogen-cold hydrazine device is required to remove CO2.

[0058] Figure 3 (a)-3(b) are the peak diagrams of carbon and nitrogen stable isotopes of the derivatized SMX monomer in this embodiment. Figure 3 (a) indicates that the carbon isotope peak of the target compound appears around 1137s; from Figure 3 (b) It can be seen that the nitrogen isotope peak of the target compound appears at around 1162 s. The results show that the detection method of this embodiment can determine the stable carbon and nitrogen isotope ratio of the derivatized SMX monomer, and the obtained peak shape is good with no impurity peaks.

[0059] Example 3

[0060] This embodiment mainly examines the method detection limit for carbon and nitrogen stable isotope analysis of derivatized SMX monomers.

[0061] (1) Weigh 20 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute with methanol to the mark to prepare a 2000 mg / L SMX stock solution. Dilute it to 1 mL to obtain a 10–500 mg / L SMX methanol solution; then, follow steps S10–S30 of Example 2 to perform derivatization reaction and monomeric carbon stable isotope analysis.

[0062] (2) Weigh 30 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute with methanol to the mark to prepare a 3000 mg / L SMX stock solution. Dilute it to 1 mL to obtain a 200–2000 mg / L SMX methanol solution; then, follow steps S10–S30 of Example 2 to perform derivatization reaction and monomeric nitrogen stable isotope analysis.

[0063] δ of derivatized SMX at different SMX concentrations 13 C value, δ 15 The N value and corresponding amplitude are as follows: Figure 4 As shown in (a)-4(b), the method detection limits for monomeric carbon and nitrogen stable isotope analysis are 30 mg / L and 400 mg / L, respectively, which are equivalent to the injection of 0.12 μg SMX and 1.6 μg SMX.

[0064] CO2 with a mass-to-charge ratio of 44 + Ions and N2 with a mass-to-charge ratio of 28 + The ion amplitude increases linearly with increasing SMX injection volume. Throughout the isotope analysis, the δ¹⁸O₂ value of derivatized SMX... 13 C value and δ 15 A deviation of N value within ±0.5‰ is considered acceptable. To ensure that differences in SMX injection volume do not interfere with the δ-value caused by isotope fractionation in actual sample detection... 13 C value and δ 15 Changes in N value, Figure 4 The range of the dashed lines in (a)-4(b) is δ 13 C value and δ 15 For reaction concentrations with N value deviations within ±0.5‰, the method detection limits for monomeric carbon and nitrogen stable isotope analysis are 150 mg / L and 600 mg / L, respectively, which is equivalent to injecting 0.6 μg SMX and 2.4 μg SMX.

[0065] Example 4

[0066] This embodiment primarily examines the precision and accuracy of carbon and nitrogen stable isotope analysis of derivatized SMX monomers.

[0067] (1) Weigh 20 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol to prepare a 2000 mg / L SMX stock solution. Dilute it to 1 mL to obtain a 200–1400 mg / L SMX methanol solution;

[0068] (2) Perform the derivatization reaction according to steps S10 and S20 of Example 2;

[0069] (3) Samples were injected multiple times within a month, and the carbon and nitrogen stable isotopes of the monomer were detected according to step S30 of Example 2.

[0070] (4) The carbon and nitrogen stable isotope ratio of the SMX standard was detected by EA-IRMS and compared and corrected with the carbon and nitrogen stable isotope ratio of the derivatized SMX solution monomer detected by GC-C-IRMS.

[0071] This embodiment involves multiple injections within one month, demonstrating good δ-ray performance. 13 C value and δ 15 N-value reproducibility. For example... Figure 5 As shown in (a)-5(b), the dashed line in the figure represents δ within the acceptable deviation range of ±0.5‰. 13 C value and δ 15 N value, δ 13 The average value of C is -36.12‰, and the δ value of 100% (18 / 18) is... 13 The C value is within the range of -36.12‰ ± 0.5‰; δ 15 The average N value was -3.42‰, and 94% (16 / 17) of the δ values ​​were... 15 The N value is within the range of -3.42‰ ± 0.5‰. The δ of the SMX standard... 13 average C and δ 15 The average values ​​of N were -27.70‰ and -2.55‰, respectively.

[0072] Figure 5 In (a)-5(b), the solid line represents the δ value of the SMX standard within the acceptable ±0.5‰ deviation range. 13 C value and δ 15 The N value. Because the derivatization reaction introduces a methyl group into SMX, the δ value of the SMX standard and the derivatized SMX... 13 The C value deviates significantly. The difference lies in the δ value between the SMX standard and the derived SMX. 15 The N values ​​are relatively close because the derivatization reaction does not change the number of N atoms, δ 15 Minor deviations in the N value may be caused by the breaking of NH bonds.

[0073] Example 5

[0074] The basic content of this embodiment is the same as that of Embodiment 2, except that: this embodiment mainly examines the effect of the amount of derivatization reagent TMSD on the detection method of carbon and nitrogen stable isotopes of sulfamethoxazole monomer, including the following steps:

[0075] S10. Weigh 20 mg of SMX standard and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with methanol to prepare a 2000 mg / L SMX stock solution. Dilute the stock solution to 1 mL to prepare SMX methanol solutions with concentrations of 200 mg / L and 1400 mg / L. Add the derivatization reagent TMSD to the above SMX methanol solutions. The molar ratio of TMSD to SMX is 9:1, 19:1, 38:1, 57:1, 76:1, and 95:1. Incubate the derivatization reaction in a 60 °C oven in the dark for 120 min.

[0076] After the derivatization reaction was completed, the methanol solvent was dried under a steady nitrogen flow. It was then reconstituted with 1 mL of acetone and vortexed for 1 min to completely dissolve it. The solution was then passed through a 0.22 μm organic filter membrane and transferred to a 2 mL brown sample vial to obtain the derivatized SMX acetone solution, which was stored at -20 °C for further analysis.

[0077] S30. Perform stable isotope analysis of carbon and nitrogen monomers according to step S30 described in Example 2.

[0078] like Figure 6 As shown, when the molar ratio of TMSD to SMX is 9:1 and 19:1, SMX cannot be completely derivatized, and obvious impurity peaks appear in its GC-C-IRMS spectrum, such as... Figure 7 As shown. When the molar ratio of TMSD to SMX is 57:1, 76:1, and 95:1, the derivatized SMX exhibits stable δ 13 C value and δ 15 The N value indicates that the SMX derivatization reaction is complete at this point and does not affect subsequent experimental measurements.

[0079] Example 6

[0080] The basic content of this embodiment is the same as that of Embodiment 2, except that this embodiment mainly examines the effect of derivatization reaction temperature on the detection method of carbon and nitrogen stable isotopes of sulfamethoxazole monomer, including the following steps:

[0081] S10. Weigh 20 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol to prepare a 2000 mg / L SMX stock solution, and dilute it to 1 mL to obtain a 200 mg / L SMX methanol solution; add the derivatization reagent TMSD to the SMX methanol solution, with a TMSD to SMX molar ratio of 76:1, and carry out the derivatization reaction in the dark at 20℃, 40℃, 60℃ and 80℃ for 120 min.

[0082] After the derivatization reaction was completed, the methanol solvent was dried under a steady nitrogen flow. It was then reconstituted with 1 mL of acetone and vortexed for 1 min to completely dissolve it. The solution was then passed through a 0.22 μm organic filter membrane and transferred to a 2 mL brown sample vial to obtain the derivatized SMX acetone solution, which was stored at -20 °C for further analysis.

[0083] S30. Perform monomeric carbon stable isotope analysis according to step S30 described in Example 2.

[0084] Example 7

[0085] The basic content of this embodiment is the same as that of Embodiment 2, except that this embodiment mainly examines the effect of derivatization reaction time on the detection method of carbon and nitrogen stable isotopes of sulfamethoxazole monomer, including the following steps:

[0086] S10. Weigh 20 mg of SMX standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol to prepare a 2000 mg / L SMX stock solution, and dilute it to 1 mL to obtain a 200 mg / L SMX methanol solution; add the derivatization reagent TMSD to the SMX standard solution, with a TMSD to SMX molar ratio of 76:1, and incubate in a 40 °C oven in the dark for derivatization reaction at 15 min, 30 min, 60 min, and 120 min.

[0087] After the derivatization reaction was completed, the methanol solvent was dried under a steady nitrogen flow. It was then reconstituted with 1 mL of acetone and vortexed for 1 min to completely dissolve it. The solution was then passed through a 0.22 μm organic filter membrane and transferred to a 2 mL brown sample vial to obtain the derivatized SMX acetone solution, which was stored at -20 °C for further analysis.

[0088] S30. Perform monomeric carbon stable isotope analysis according to step S30 described in Example 2.

[0089] like Figure 8 As shown in (a)-8(b), due to the δ during the derivatization reaction... 13 The C value varies significantly, therefore the δ of the derived SMX is used. 13 The variation characteristics of C value are used to optimize reaction temperature and reaction time (the dashed line in the figure represents the deviation of ±0.5‰).

[0090] exist Figure 8 In (a), the derivatization method of the present invention exhibits stable δ values ​​at reaction temperatures ranging from 20°C to 80°C. 13 The C value indicates δ 13 The C value is not affected by the reaction temperature. Furthermore, since room temperature varies considerably with the seasons, this example investigates the δ value at different reaction times when the oven temperature is 40°C. 13 The C value. Figure 8 In (b), a stable δ value was observed for reaction times ranging from 15 to 120 min. 13 The C value indicates that the derivatization reaction of the present invention can be completed within 15 minutes.

[0091] The present invention and its embodiments have been described above illustratively. This description is not restrictive, and the data used is only one embodiment of the present invention. The actual combination of data is not limited to this. Therefore, if those skilled in the art are inspired by this description and, without departing from the spirit of the present invention, devise similar embodiments and examples of the technical solution without creative design, all such embodiments and examples should fall within the protection scope of the present invention.

Claims

1. A method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer, characterized in that: Includes the following steps: S10. Sulfamethoxazole is dissolved in a first solvent to prepare a solution of sulfamethoxazole in the first solvent. Trimethylsilyldiazomethane is used as a derivatizing agent to derivatize the solution of sulfamethoxazole in the first solvent to obtain a derivatized solution of sulfamethoxazole in the first solvent. The molar ratio of trimethylsilyldiazomethane to sulfamethoxazole is (57-95):

1. S20. Remove the first solvent and redissolve the derivatized sulfamethoxazole in the second solvent to obtain a solution of derivatized sulfamethoxazole in the second solvent. S30. The derivatized sulfamethoxazole solution in the second solvent is separated by gas chromatography. The gas chromatograph has an injection port temperature of 250℃, helium as the carrier gas at a flow rate of 1 mL / min, and an injection volume of 4 μL. Splitless injection is used. The gas chromatograph temperature program is as follows: initial temperature 80℃, hold for 2 min; increase to 180℃ at 30℃ / min, hold for 2 min; increase to 280℃ at 10℃ / min, hold for 5 min; preparation run time is 10 min; then it is burned in a combustion furnace to convert into CO2 and N2. The carbon stable isotope ratio and nitrogen stable isotope ratio are determined by IRMS.

2. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: The first solvent is methanol; the second solvent is one or more of acetone, n-hexane, and methanol.

3. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: In step S10, the reaction time of the derivatization reaction is 15 to 120 minutes.

4. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: In step S10, the reaction temperature of the derivatization reaction is 20℃~80℃.

5. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: In step S10, the concentration of the sulfamethoxazole solution in the first solvent is 200-2000 mg / L.

6. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: The specific process of step S30 is as follows: the derivatized sulfamethoxazole solution in the second solvent is injected twice into the gas chromatograph for separation, and then enters the combustion furnace to be converted into CO2 and N2. The carbon stable isotope ratio and nitrogen stable isotope ratio are determined by IRMS, respectively. Before determining the nitrogen stable isotope ratio by IRMS, the CO2 converted by combustion in the combustion furnace is removed by liquid nitrogen and hydrazine.

7. The method for detecting the carbon and nitrogen stable isotopes of sulfamethoxazole monomer according to claim 1, characterized in that: In step S30, the temperature in the combustion furnace is 1000℃~1100℃.

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