Extraction and detection method of porphyrin compound

Abstract

The invention relates to a porphyrin compound extraction and detection method. The extraction method comprises the following steps: weighing a sample to be detected, adding an organic solvent A containing a reagent B and a reagent C, heating and refluxing, and cooling to room temperature to obtain a mixture 1; performing ultrasonic oscillation on the mixture 1 by using ultrapure water and a reagent D in sequence to obtain a mixture 2; centrifuging the mixture 2 to separate out an organic phase and a water phase; the water phase is subjected to alkalization treatment, organic compounds are extracted from the water phase, and the free porphyrin component is obtained. According to the method, the problems of low porphyrin compound extraction efficiency, serious environmental pollution, difficulty in eliminating environmental matrix interference and the like in the prior art are solved, and efficient, environment-friendly and accurate enrichment and detection of the porphyrin compounds are realized.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of environmental analytical chemistry and geochemistry, and specifically relates to the selective extraction and trace detection technology of porphyrin compounds in complex environmental matrices, and is particularly suitable for environmental samples such as soil and sediments with high humus content. Background Art

[0002] As degradation products of biomacromolecules such as chlorophyll and heme, porphyrin compounds are important biomarkers for studying carbon and nitrogen cycles and paleoenvironmental reconstruction (Sinninghe Damsté et al., 2019, Organic Geochemistry). Their occurrence forms and concentration distribution in soil directly reflect microbial activity and organic matter conversion efficiency (Lü et al., 2020, Geochimica et Cosmochimica Acta). However, due to the interference of complex environmental matrices and the trace characteristics of target substances (ng / g level), existing technologies are difficult to achieve high-fidelity extraction and accurate detection, which has become a key bottleneck restricting environmental geochemical research.

[0003] The current mainstream liquid-liquid extraction (LLE) and ultrasound-assisted extraction (UAE) have significant defects: (1) The solvent consumption is as high as 20-50 mL / g sample (Wang et al., 2018, Analytical Chemistry), which increases the processing cost and produces VOCs pollution; (2) Co-extracts such as humic acid (HA) and metal complexes account for more than 60% (Zhang et al., 2021, Journal of Chromatography A), resulting in matrix inhibition effect (Matrix Effect) in subsequent detection; (3) The existing method performs porphyrin protonation extraction under acidic conditions (pH 2-3), but the strong acid environment causes demetallation reaction of tetrapyrrole macrocycles, especially causing irreversible damage to Mg-porphyrin (degradation rate is as high as 65%), which seriously affects the accuracy of paleoenvironmental reconstruction data (Petersen et al., 2021, Geochimica et Cosmochimica Acta).

[0004] The sensitivity bottlenecks of existing detection methods: (1) Although high performance liquid chromatography (HPLC) coupled with a UV detector is widely used, it faces the following problems: irreversible adsorption of humic acid fragments on the C18 column, resulting in a 25% attenuation of column efficiency (Chen et al., 2020, Environmental Science & Technology); and the UV spectrum is interfered by carotenoids near 400 nm, reducing the signal-to-noise ratio (S / N) to 2.1-3.5 (Guo et al., 2021, Microchemical Journal); (2) Although high-resolution mass spectrometry (HRMS) has achieved a detection limit of ng / L, humic acid derivatives produce a strong matrix effect in the electrospray ionization source (signal suppression rate 78-92%), forcing the sample to be diluted 10-100 times, resulting in the inability to detect low-abundance porphyrins (Kujawinski et al., 2022, Analytical Chemistry). Summary of the invention

[0005] To solve the above problems, the present invention aims to provide a method for extracting and detecting porphyrin compounds, so as to solve the problems existing in the prior art such as low extraction efficiency, severe environmental pollution, and difficulty in eliminating environmental matrix interference, and to achieve efficient, environmentally friendly, and accurate enrichment and detection of porphyrin compounds.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for extracting porphyrin compounds, comprising the following steps:

[0008] Step 1: weigh a sample to be tested, add an organic solvent A containing reagent B and reagent C, heat to reflux, and cool to room temperature to obtain a mixture 1;

[0009] Wherein, the organic solvent A is selected from one or more of toluene, xylene, dichloromethane, chloroform, and carbon disulfide;

[0010] Reagent B is selected from one or more of ethanol, isopropanol, and propylene glycol, and reagent C is selected from one or more of tranexamic acid, methanesulfonyl chloride, dimethyl sulfoxide, methanesulfonic acid, and toluenesulfonic acid.

[0011] Step 2: Ultrapure water and reagent D are sequentially used to ultrasonically oscillate the mixture 1 to obtain a mixture 2;

[0012] Reagent D is a small molecule hydrocarbon solvent with a carbon number not exceeding 8, for example, it can be n-hexane and / or n-heptane.

[0013] Step 3: Centrifuge the mixture 2 to separate the organic phase and the aqueous phase; alkalize the aqueous phase, extract the organic compounds from the aqueous phase, and obtain the free porphyrin component.

[0014] In step 1 of the present invention, reagent A and a small amount of reagent B are used to extract organic matter in soil. Reagent C reacts with metal porphyrin in the extracted organic matter to undergo a demetallization reaction; in step 2, water and reagent D are ultrasonically vibrated to prepare for centrifugation; in step 3, centrifugation and separation are performed to obtain an organic phase and an aqueous phase; then NaOH is added to the aqueous phase to neutralize excess reagent C, and then reagent A is used to extract the target, i.e., the free porphyrin component.

[0015] Furthermore, the sample to be tested is soil, sediment, coal or any other sample rich in humus.

[0016] Furthermore, when the sample to be tested is soil, a pretreatment step is also included; the pretreatment includes the steps of air-drying and grinding the soil.

[0017] Furthermore, the air-drying treatment is carried out at a temperature of 35±2°C for at least 24 hours. If the temperature exceeds this range, the soil components will be destroyed and the dried soil sample will lose its value. If the temperature is too low, the air-drying effect cannot be achieved. Preferably, a fine drying oven is used, and the air-drying time of the soil sample is 24 hours to complete a batch of drying. The hot air used to dry the soil sample is purified and adsorbed, and it is a constant heat flow.

[0018] Furthermore, the grinding is to grind the soil particles in a clockwise direction using an agate mortar, and extract soil particles with a particle size of less than 100 meshes as samples to be tested.

[0019] Further, the sample to be tested is 3-20 g; the dosage of reagent A is 20-50 mL; the dosage of reagent B is 2-25 mL; the dosage of reagent C is 2-25 mL;

[0020] The present application improves extraction efficiency and analysis accuracy by optimizing the dosage of reagents. In addition, the sample to be tested can be processed according to the above ratio and is not limited to the weight range of the sample to be tested.

[0021] Further, after adding organic solvent A to the sample to be tested, heating and reflux are maintained at 100-200° C. for 1-5 hours, and then the temperature is increased to 150-200° C. and maintained for another 2-8 hours;

[0022] Furthermore, the ultrasonic oscillation time in step 2 is 5-30 minutes;

[0023] Furthermore, the centrifugation speed in step 3 is 3000-10000 rpm, and is repeated 3-5 times;

[0024] Furthermore, the alkalization treatment in step 3 is to add sodium hydroxide and / or potassium hydroxide particles to the aqueous phase until no bubbles appear in the mixed solution;

[0025] Furthermore, the step 3 of extracting the organic compound from the aqueous phase is to add reagent A to the aqueous phase for extraction;

[0026] Furthermore, the method for extracting porphyrin compounds comprises the following steps:

[0027] Step 1: weigh a sample to be tested, add an organic solvent A containing reagent B and reagent C, heat and reflux, maintain at 100-200° C. for 1-5 hours, then increase the temperature to 150-200° C. and maintain for another 2-8 hours, and then cool to room temperature to obtain a mixture 1; the sample to be tested is soil, sediment, coal or any other sample rich in humus;

[0028] Wherein, the organic solvent A is selected from one or more of toluene, xylene, dichloromethane, chloroform, and carbon disulfide;

[0029] Reagent B is selected from one or more of ethanol, isopropanol, and propylene glycol, and reagent C is selected from one or more of tranexamic acid, methanesulfonyl chloride, dimethyl sulfoxide, methanesulfonic acid, and toluenesulfonic acid;

[0030] The sample to be tested is 3-20g; the dosage of reagent A is 20-50mL; the dosage of reagent B is 2-25mL; the dosage of reagent C is 2-25mL;

[0031] Step 2: Ultrapure water and reagent D are sequentially used to ultrasonically oscillate mixture 1 for 5-30 min to obtain mixture 2;

[0032] Reagent D is a small molecule hydrocarbon solvent with a carbon number not exceeding 8.

[0033] Step 3: centrifuge the mixture 2 at a speed of 3000-10000 rpm, repeat 3-5 times, and separate the organic phase and the aqueous phase; add sodium hydroxide and / or potassium hydroxide particles to the aqueous phase for alkalization until no bubbles appear in the mixed solution; add reagent A to the aqueous phase to extract the organic compound to obtain a free porphyrin component.

[0034] In a second aspect, the present invention provides a method for detecting porphyrin compounds, the detection method comprising: extracting porphyrin compounds from a sample to be tested according to the method described in the first aspect, and then detecting the porphyrin compounds.

[0035] Furthermore, the detection method is to use a positive ion electrospray ionization source combined with a high-resolution mass spectrometer for detection.

[0036] Specifically, the extracted free porphyrin component is subjected to high-resolution mass spectrometry analysis using the electrospray ionization source (ESI) positive ion mode (+) of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), wherein the mass resolution of the high-resolution mass spectrometry analysis at m / z 400 is greater than 280,000.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The invention significantly improves the extraction efficiency of porphyrin compounds by optimizing the extraction reagents and conditions, reduces the usage of organic solvents, and reduces costs and environmental pollution.

[0039] The present invention adopts a positive ion electrospray ionization source combined with a high-resolution mass spectrometer for detection, which can effectively eliminate the interference of complex environmental matrices, realize accurate enrichment and detection of porphyrin compounds, and improve the accuracy and reliability of the test results. The method provided by the present invention directly measures the content of free porphyrin compounds in humus-rich samples such as soil for the first time, and can effectively process porphyrin compound samples with different content levels, and the porphyrin compound content range covers 0.01wt%-100wt%.

[0040] The present invention facilitates researchers to quickly and accurately obtain molecular composition information of porphyrin compounds, and provides a powerful tool for in-depth research on chemical processes and biogeochemical cycles in soil environments. In addition, the method provided by the present invention has a wide range of applications, not only for soil samples, but also for other humus-rich samples such as sediments and coal, and has good versatility and practicality.

[0041] In summary, the present invention achieves breakthroughs through the following core designs:

[0042] (1) Composite solvent system: The synergistic effect of organic solvent A and reagent B / C forms a polarity gradient (ε decreases from 25.3 to 18.7), selectively dissolving porphyrin and precipitating humic acid (precipitation efficiency > 95%);

[0043] (2) Mild alkalinization strategy: Control the pH to 8.5-9.0 to stabilize the metalloporphyrin (dissociation rate <8%) and release free porphyrin at the same time;

[0044] (3) Ultra-high mass resolution mass spectrometry detection: The extracted free porphyrin components were subjected to high-resolution mass spectrometry detection using the electrospray ionization source (ESI) of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in positive ion mode (+). The mass resolution at m / z 400 was greater than 280,000. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1It is a flow chart of the porphyrin compound detection method provided by the present invention;

[0046] Figure 2 The high-resolution mass spectrum and the corresponding carbon number and double bond equivalent number (DBE) distribution diagram of the porphyrin compounds are obtained by analyzing the porphyrin compounds in farmland soil using the positive ion electrospray ionization mode combined with Fourier transform ion cyclotron resonance mass spectrometry in Example 3. DETAILED DESCRIPTION

[0047] The existing technology has problems such as low extraction efficiency, serious environmental pollution, and difficulty in eliminating environmental matrix interference. After intensive research, the inventors of the present invention have proposed a method for extracting and detecting porphyrin compounds to achieve efficient, environmentally friendly, and accurate enrichment and detection of porphyrin compounds.

[0048] In a first aspect, the present invention provides a method for extracting porphyrin compounds, comprising the following steps:

[0049] Step 1: weigh a sample to be tested, add an organic solvent A containing reagent B and reagent C, heat to reflux, and cool to room temperature to obtain a mixture 1;

[0050] Wherein, the organic solvent A is selected from one or more of toluene, xylene, dichloromethane, chloroform, and carbon disulfide;

[0051] Reagent B is selected from one or more of ethanol, isopropanol, and propylene glycol, and reagent C is selected from one or more of tranexamic acid, methanesulfonyl chloride, dimethyl sulfoxide, methanesulfonic acid, and toluenesulfonic acid.

[0052] Step 2: Ultrapure water and reagent D are sequentially used to ultrasonically oscillate the mixture 1 to obtain a mixture 2;

[0053] Reagent D is a small molecule hydrocarbon solvent with a carbon number not exceeding 8, for example, it can be n-hexane and / or n-heptane.

[0054] Step 3: Centrifuge the mixture 2 to separate the organic phase and the aqueous phase; alkalize the aqueous phase, extract the organic compounds from the aqueous phase, and obtain the free porphyrin component.

[0055] As a preferred embodiment, the sample to be tested is soil, sediment, coal or any other sample rich in humus.

[0056] As a preferred embodiment, when the sample to be tested is soil, it also includes a pretreatment step; the pretreatment includes the steps of air-drying and grinding the soil.

[0057] As a preferred embodiment, the air-drying treatment is carried out at a temperature of 35±2°C (the temperature may be 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C or any value within the range), and the air-drying treatment time is at least 24 hours (for example, it may be 24h, 25h, 26h, 27h, 28h, 29h, 30h, 36h, 48h, 60h, 72h, etc.). If this temperature range is exceeded, the soil components will be destroyed, and the dried soil sample will lose its value. If the temperature is too low, the air-drying effect will not be achieved. Preferably, a fine drying box is used, and the air-drying time of the soil sample is 24 hours to complete a batch of drying. The hot air of the dried soil sample is purified and adsorbed, and it is a constant heat flow.

[0058] As a preferred embodiment, the grinding is to grind the soil particles in a clockwise direction using an agate mortar, and extract soil particles with a particle size of less than 100 mesh as the sample to be tested.

[0059] As a preferred embodiment, the sample to be tested is 3-20g; the amount of reagent A is 20-50mL (for example, it can be 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL or any value within the range); the amount of reagent B is 2-25mL (for example, it can be 5mL, 10mL, 15mL, 20mL, 25mL or any value within the range); the amount of reagent C is 2-25mL (for example, it can be 5mL, 10mL, 15mL, 20mL, 25mL or any value within the range);

[0060] The present application improves extraction efficiency and analysis accuracy by optimizing the dosage of reagents. In addition, the sample to be tested can be processed according to the above ratio and is not limited to the weight range of the sample to be tested.

[0061] As a preferred embodiment, the organic solvent A is added to the sample to be tested, and then heated to reflux, and maintained at 100-200° C. (for example, it can be 100° C., 110° C., 120° C., 130° C., 140° C., 150° C., 160° C., 170° C., 180° C., 190° C., 200° C. or any value within the range) for 1-5 hours (for example, it can be 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h or any value within the range). value), then raising the temperature to 150-200°C (for example, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C, 200°C or any value within the range) and maintaining for 2-8 hours (for example, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h or any value within the range);

[0062] As a preferred embodiment, the ultrasonic oscillation time in step 2 is 5-30 min (for example, it can be 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value within the range);

[0063] As a preferred embodiment, the centrifugal speed in step 3 is 3000-10000 rpm (for example, it can be 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm or any value within the range), and is repeated 3-5 times (for example, it can be 3 times, 4 times or 5 times);

[0064] As a preferred embodiment, the alkalization treatment in step 3 is to add sodium hydroxide and / or potassium hydroxide particles to the aqueous phase until no bubbles appear in the mixed solution;

[0065] As a preferred embodiment, the step of extracting the organic compound from the aqueous phase in step 3 is to add reagent A to the aqueous phase for extraction;

[0066] As a preferred embodiment, the method for extracting porphyrin compounds comprises the following steps:

[0067] Step 1: weigh a sample to be tested, add an organic solvent A containing reagent B and reagent C, heat and reflux, maintain at 100-200° C. for 1-5 hours, then increase the temperature to 150-200° C. and maintain for another 2-8 hours, and then cool to room temperature to obtain a mixture 1; the sample to be tested is soil, sediment, coal or any other sample rich in humus;

[0068] Wherein, the organic solvent A is selected from one or more of toluene, xylene, dichloromethane, chloroform, and carbon disulfide;

[0069] Reagent B is selected from one or more of ethanol, isopropanol, and propylene glycol, and reagent C is selected from one or more of tranexamic acid, methanesulfonyl chloride, dimethyl sulfoxide, methanesulfonic acid, and toluenesulfonic acid;

[0070] The sample to be tested is 3-20g; the dosage of reagent A is 20-50mL; the dosage of reagent B is 2-25mL; the dosage of reagent C is 2-25mL;

[0071] Step 2: Ultrapure water and reagent D are sequentially used to ultrasonically oscillate mixture 1 for 5-30 min to obtain mixture 2;

[0072] Reagent D is a small molecule hydrocarbon solvent with a carbon number not exceeding 8.

[0073] Step 3: centrifuge the mixture 2 at a speed of 3000-10000 rpm, repeat 3-5 times, and separate the organic phase and the aqueous phase; add sodium hydroxide and / or potassium hydroxide particles to the aqueous phase for alkalization until no bubbles appear in the mixed solution; add reagent A to the aqueous phase to extract the organic compound to obtain a free porphyrin component.

[0074] In a second aspect, the present invention provides a method for detecting porphyrin compounds, the detection method comprising: extracting porphyrin compounds from a sample to be tested according to the method described in the first aspect, and then detecting the porphyrin compounds.

[0075] As a preferred embodiment, the detection method is to use a positive ion electrospray ionization source combined with a high-resolution mass spectrometer for detection.

[0076] Specifically, the extracted free porphyrin component is subjected to high-resolution mass spectrometry analysis using the electrospray ionization source (ESI) positive ion mode (+) of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), wherein the mass resolution of the high-resolution mass spectrometry analysis at m / z 400 is greater than 280,000.

[0077] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0078] In the specific embodiment of the present invention, the Solari 2XR ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) produced by Bruker is used. The operating conditions of mass spectrometry detection are: using a Bruker Solari 2XR ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) equipped with a 7.0T superconducting magnet and an electrospray ion source. The standard Bruker electrospray ionization source positive ion ionization mode (+ESI) is used as the ionization method, the injection rate is 180 μL / h, the electrospray voltage is 5.0 kV, the end plate bias voltage is -500 V, the nebulizing gas pressure is 2.0 bar, the drying gas temperature is 250°C, and the flow rate is 5.0 L / min. Mass spectrometry data were collected in broadband mode (150–1000 m / z), with a transient size of 4 M, an FID signal period of 1.4230 s, an ion accumulation time of 0.030 s, a flight time of 0.750 ms, and a total ion current (TIC) intensity of 5.0 × 10 8 -1.0×10 9 between.

[0079] Example 1

[0080] In this embodiment, the samples were collected from the corn and wheat rotation experimental field of the National Field Scientific Observation and Research Station of the Key Earth Zone along the Bohai Sea Coast in Tianjin (38°56′57″N, 117°33′39″E). The experimental field was sown with the Weike 702 corn variety (Zhengzhou Weike Crop Breeding Technology Co., Ltd., Henan Jinyuan Seed Co., Ltd.) in June 2024 and harvested in October 2024. After harvesting, surface soil samples were collected separately. Multiple topsoil samples (0-5cm) were randomly taken and mixed in the sampling area according to the serpentine sampling method. After the samples were collected, they were immediately bagged, sealed and taken back to the laboratory for air drying, grinding and sieving.

[0081] Pretreatment of samples to be tested:

[0082] 1) Soil air drying: When air drying soil samples, the allowable temperature is 35℃±2℃. Use a precision drying oven and the soil samples should be air dried for 24 hours.

[0083] 2) Grinding and screening: Grind the soil particles in a clockwise direction using an agate mortar and extract soil particles smaller than 100 mesh as the test samples.

[0084] This example selected the surface soil of the corn rotation field because its humus layer is rich in plant residue degradation products (such as chlorophyll derivatives) and microbial metabolites, providing an ideal matrix for the enrichment of porphyrin compounds (such as pheophorbide and heme derivatives). By controlling the air-drying temperature (<40°C) and the grinding particle size (<150μm), the oxidation loss of the porphyrin structure can be effectively reduced, and the specific surface area can be increased to improve the subsequent extraction efficiency (verified by comparative experiments, the extraction rate of 100 mesh samples is 23.6%±2.1% higher than that of 60 mesh samples).

[0085] Example 2

[0086] This embodiment includes the following steps:

[0087] Step 1: Weigh 20 g of soil sample and place it in a filter paper tube of a Soxhlet extractor. Add 50 mL of toluene, 10 mL of isopropanol and 5 mL of toluenesulfonic acid into the extraction bottle in sequence and heat to reflux; keep the mixture at 120°C for 2 hours, then increase the temperature to 150°C and keep the temperature for another 4 hours, and then cool to room temperature to obtain a mixture 1.

[0088] Step 2: The mixture 1 was ultrasonically oscillated with 30 mL of ultrapure water and 20 mL of n-heptane in sequence, with each oscillation time being 15 min, to obtain a mixture 2.

[0089] Step 3: High-speed centrifugation of mixture 2 at a speed of 5000 rpm was repeated three times to separate the organic phase and the aqueous phase; sodium hydroxide particles were slowly added to the aqueous phase for alkalization until no bubbles appeared in the mixed solution; then 10 mL of toluene reagent was added to extract organic compounds from the aqueous phase to obtain free porphyrin components.

[0090] Example 3

[0091] The detection method of porphyrin compounds comprises the following steps:

[0092] Step S1, mass spectrometry analysis: the free porphyrin component is dissolved with toluene and methanol to adjust the injection concentration, and the separated free porphyrin component is subjected to high-resolution mass spectrometry analysis using an electrospray ionization source (ESI) in a positive ion mode (+) of a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), wherein the mass resolution of the FT-ICR MS at m / z 400 is greater than 280,000.

[0093] Step S2, spectrum analysis: After the sample test is completed, the reference mass table is used for recalibration. After internal calibration (by detecting the standard calibration solution, the mass calibration is performed according to the reference mass table to achieve the purpose of calibrating the instrument mass accuracy), the mass error in the entire mass range is ensured to be controlled within 600ppb, and the mass spectrum peak with a signal-to-noise ratio higher than 6 is selected. The correct chemical formula is assigned to the compound based on the specific atomic number of carbon atoms, hydrogen atoms, oxygen atoms, nitrogen atoms and sulfur atoms. After the chemical formula assignment is completed, a detailed list of molecular masses and chemical formulas will be obtained for further analysis and research. Among them, the number of carbon atoms is 1-80, the number of hydrogen atoms is 2-120, the number of oxygen atoms is 0-25, the number of nitrogen atoms is 0-3, and the number of sulfur atoms is 0-2.

[0094] Step S3, data processing: convert the mass list obtained in step S2 into a table, and divide it into different categories for analysis, and then perform data analysis through visual data processing. Specifically, the target component is subjected to high-resolution mass spectrometry analysis to obtain the spectrum of the porphyrin compound. After spectrum analysis and data processing, a more intuitive carbon number-equivalent double bond diagram is obtained. The horizontal axis of the diagram is the carbon number in the molecular formula of the compound, the vertical axis is the equivalent double bond number of the compound, and the point size represents the signal intensity. Figure 2 In the high-resolution mass spectrum of the enriched free porphyrin compounds, each mass spectrum peak represents a specific porphyrin compound. These peaks correspond to different mass-to-charge ratios (m / z), showing the ion signal intensity of the porphyrin compounds, indicating that the porphyrin compounds have been successfully detected; the corresponding distribution diagram of carbon number and double bond equivalent number (DBE) of porphyrin compounds shows the relative abundance of porphyrin compounds with different DBE values, which indicates the diversity and complexity of porphyrin compounds, further proving the feasibility of this method for the detection of porphyrin compounds.

[0095] In summary, the present invention achieves breakthroughs through the following core designs:

[0096] (1) Composite solvent system: The synergistic effect of organic solvent A and reagent B / C forms a polarity gradient (ε decreases from 25.3 to 18.7), selectively dissolving porphyrin and precipitating humic acid (precipitation efficiency > 95%);

[0097] (2) Mild alkalinization strategy: Control the pH to 8.5-9.0 to stabilize the metalloporphyrin (dissociation rate <8%) and release free porphyrin at the same time;

[0098] (3) Ultra-high mass resolution mass spectrometry detection: The extracted free porphyrin components were subjected to high-resolution mass spectrometry detection using the electrospray ionization source (ESI) of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in positive ion mode (+). The mass resolution at m / z 400 was greater than 280,000.

[0099] The method provided by the present invention significantly improves the extraction efficiency of porphyrin compounds by optimizing extraction reagents and conditions, reduces the amount of organic solvents used, and reduces costs and environmental pollution. The present invention adopts a positive ion electrospray ionization source in combination with a high-resolution mass spectrometer for detection, can effectively eliminate the interference of complex environmental matrix, achieve accurate enrichment and detection of porphyrin compounds, and improve the accuracy and reliability of test results. In addition, the present invention provides detailed mass spectrometry and data processing methods, so that the analysis process is more systematic and standardized, and it is convenient for researchers to quickly and accurately obtain the molecular composition information of porphyrin compounds, and provide a powerful tool for in-depth study of chemical processes and biogeochemical cycles in soil environments. The present invention has a wide range of applications, is not only applicable to soil samples, but also can be used for other humus-rich samples such as sediments and coal, and has good versatility and practicality.

[0100] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting porphyrin compounds, characterized in that: The following steps are involved: Step 1: weigh a sample to be tested, add an organic solvent A containing reagent B and reagent C, heat to reflux, and cool to room temperature to obtain a mixture 1; Wherein, the organic solvent A is selected from one or more of toluene, xylene, dichloromethane, chloroform, and carbon disulfide; Reagent B is selected from one or more of ethanol, isopropanol, and propylene glycol, and reagent C is selected from one or more of tranexamic acid, methanesulfonyl chloride, dimethyl sulfoxide, methanesulfonic acid, and toluenesulfonic acid. Step 2: Ultrapure water and reagent D are sequentially used to ultrasonically oscillate the mixture 1 to obtain a mixture 2; Reagent D is a small molecule hydrocarbon solvent with a carbon number not exceeding 8. Step 3: Centrifuge the mixture 2 to separate the organic phase and the aqueous phase; alkalize the aqueous phase, extract the organic compounds from the aqueous phase, and obtain the free porphyrin component.

2. The method for extracting porphyrin compounds according to claim 1, characterized in that: The sample to be tested is any one of soil, sediment or coal, or a combination of at least two of them.

3. The method for extracting porphyrin compounds according to claim 2, characterized in that, When the sample to be tested is soil, a pre-treatment step is also included; the pre-treatment includes the steps of air-drying and grinding the soil; The air-drying treatment is carried out at a temperature of 35±2°C for at least 24 hours; The grinding is to grind the soil particles in a clockwise direction, and extract soil particles with a particle size of less than 100 meshes as samples to be tested.

4. The method for extracting porphyrin compounds according to claim 3, characterized in that: The sample to be tested is 3-20 g; the dosage of reagent A is 20-50 mL; the dosage of reagent B is 2-25 mL; and the dosage of reagent C is 2-25 mL.

5. The method for extracting porphyrin compounds according to claim 4, characterized in that: After adding organic solvent A to the sample to be tested, heat and reflux, keep it at 100-200°C for 1-5 hours, then increase the temperature to 150-200°C and keep it for another 2-8 hours.

6. The method for extracting porphyrin compounds according to claim 1 or 2, characterized in that: The ultrasonic oscillation time in step 2 is 5-30 minutes.

7. The method for extracting porphyrin compounds according to claim 1 or 2, characterized in that: The centrifugal speed in step 3 is 3000-10000 rpm, and repeated 3-5 times; The alkalization treatment in step 3 is to add sodium hydroxide and / or potassium hydroxide particles to the aqueous phase until no bubbles appear in the mixed solution.

8. The method for extracting porphyrin compounds according to claim 1 or 2, characterized in that: The step 3 of extracting the organic compound from the aqueous phase is to add reagent A to the aqueous phase for extraction.

9. A method for detecting porphyrin compounds, the detection method comprising: The porphyrin compounds are extracted from the sample to be tested according to the method described in the first aspect, and then the porphyrin compounds are detected.

10. The detection method according to claim 9, characterized in that: The detection method is to use a positive ion electrospray ionization source combined with a high-resolution mass spectrometer for detection.

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