Application of aminated beta-cyclodextrin composite material and method for simultaneously analyzing endogenous and exogenous organic matters in urine by using aminated beta-cyclodextrin composite material

Abstract

The invention relates to application of an aminated beta-cyclodextrin composite material and a method for simultaneously analyzing endogenous and exogenous organic matters in urine by using the aminated beta-cyclodextrin composite material. According to the method, the cellulose-loaded aminated beta-cyclodextrin composite material is used as a solid-phase extraction agent for the first time, the endogenous markers in the urine are efficiently extracted, and the combined analysis of the endogenous markers and the exogenous markers is realized at the same time. A target compound can be efficiently extracted from a complex urine matrix through an optimized solid-phase extraction process which comprises the steps of activation, sample loading, drying, elution, nitrogen blowing and the like. Then, a liquid chromatography-mass spectrometry technology is utilized for analysis, qualitative and quantitative analysis is performed on 80 target objects in a multi-reaction monitoring (MRM) mode, and the method has good recovery rate (60%-130%) and excellent stability and repeatability.

Description

Technical Field

[0001] The present invention relates to solid phase extraction technology and instrumental analysis technology, in particular to the use of an amino-β-cyclodextrin composite material and a method for simultaneously analyzing endogenous and exogenous organic matter in urine. The technology includes a solid phase extraction pretreatment method and a liquid chromatography-mass spectrometry (LC-MS) combined analysis method, and is applicable to the fields of environmental monitoring, drug metabolite analysis, biomedical research, and clinical testing. Background Art

[0002] Urine contains a large amount of organic matter, including exogenous pollutants and endogenous biomarkers. These substances are of great significance in the fields of environmental monitoring, public health research and early diagnosis of diseases. Exogenous organic matter refers to pollutants from the external environment, which usually enter the human body through food, drinking water or air. Exogenous organic matter includes but is not limited to: plasticizers (such as phthalates, parabens, bisphenols). These chemicals are widely used in plastic products and may enter the human body through skin contact, breathing or food intake. They are closely related to endocrine disruption, immune system abnormalities and reproductive health problems; organophosphorus flame retardants, these substances are often used in furniture, electronic products and building materials. Studying their metabolites and residues in the human body will help reveal their potential hazards to the nervous system, immune system and other aspects; perfluoroalkyl substances (PFAS), these chemicals have extremely high stability and can exist in the environment for a long time, causing long-term effects on water sources, air and human health. Its potential harm to the liver, immune system and endocrine system has gradually attracted widespread attention; drugs such as antidepressants and antibiotics are usually excreted through urine in the form of parent or metabolites, reflecting the use of drugs and their effects on the human body. In addition, these drugs are excreted into the aquatic ecosystem through urine, which, on the one hand, increases the abundance of resistance genes in the ecological environment, and on the other hand, affects the normal biological rhythm of organisms, posing a major threat to ecological health; other exogenous organic matter also includes food and lifestyle indicators, such as nicotine, sorbic acid, aspartame, caffeine, etc. As common living chemicals, their metabolites in urine can be used as indicators of individual living habits and food intake, which is helpful to study the relationship between living habits and diseases. Endogenous organic matter includes biomarkers related to physical health status and disease processes, such as oxidative stress bioindicators, which reflect the level of oxidative stress in the body; DNA methylation indicators, which are closely related to a variety of diseases (such as cancer, cardiovascular disease, etc.); emotional hormones, such as cortisol, dopamine and other hormone levels, which are closely related to mental health, stress response and the occurrence of some mental illnesses; estrogen levels in different genders and age groups are related to reproductive health, metabolic diseases and endocrine disorders. Urine, as a common and easily accessible biological sample, can simultaneously reflect the metabolism of these exogenous pollutants and endogenous biomarkers. Therefore, the quantitative analysis of these substances in urine has important clinical and research value. In addition, the development of an analytical method that can simultaneously analyze exogenous and endogenous organic matter in urine is both convenient and fast and saves analysis costs.

[0003] As a natural cyclic oligosaccharide, β-cyclodextrin has unique molecular recognition ability and is widely used to adsorb a variety of compounds. Amino-modified β-cyclodextrin polymers significantly improve their hydrophilicity, adsorption capacity and selectivity for different target molecules by introducing amino groups into the cyclodextrin polymer structure. Its adsorption capacity for various organic substances is enhanced through hydrogen bonding and electrostatic effects, especially for compounds with strong polarity or charge. It is particularly suitable for the extraction of target compounds in complex matrices. β-cyclodextrin and its modified materials have achieved remarkable results in the field of water purification, but its potential in the extraction and analysis of endogenous and exogenous organic matter in urine samples has not been fully explored. In addition, liquid chromatography-mass spectrometry (LC-MS) technology has become an indispensable tool in the analysis of urine samples due to its high resolution and high sensitivity. However, traditional methods usually measure endogenous organic matter and exogenous pollutants in urine separately, which not only increases the complexity of sample pretreatment, but also increases the analysis cost. In addition, due to the complexity of the urine matrix, it is difficult for existing extraction methods to ensure high recovery while taking into account the accurate analysis of endogenous and exogenous substances. Therefore, developing a pretreatment method that is suitable for the efficient extraction of both endogenous and exogenous organic matter, combined with an optimized analytical process, is the key to solving the problem of multi-target detection in complex urine samples. Summary of the invention

[0004] The present invention aims to provide a low-cost and efficient solid phase extraction technology, combined with an optimized liquid chromatography-mass spectrometry (LC-MS / MS) analysis method, which can extract 80 organic substances from urine, including exogenous and endogenous organic substances. The present invention uses a cellulose-loaded amino-β-cyclodextrin composite material, which not only significantly improves the efficiency of solid phase extraction, but also achieves efficient separation and accurate detection of multiple organic substances in urine by optimizing liquid chromatography and mass spectrometry conditions.

[0005] To achieve the above object, the present invention provides a technical solution that uses an amino-β-cyclodextrin composite material loaded on cellulose as a solid phase extractant for simultaneously analyzing endogenous and exogenous organic matter in urine.

[0006] The second technical solution provided by the present invention is a method for simultaneously analyzing endogenous and exogenous organic matter in urine using the above-mentioned amino-β-cyclodextrin composite material loaded on cellulose as a solid phase extraction agent, comprising the following steps:

[0007] 1) Urine pretreatment;

[0008] 2) using an amino-β-cyclodextrin composite material supported on cellulose as a solid phase extractant;

[0009] 3) Extracting urine using a solid phase extraction column;

[0010] 4) performing liquid chromatography-mass spectrometry analysis on the extracted samples to detect endogenous and exogenous organic matter;

[0011] The step 1) comprises:

[0012] Prepare ammonium acetate buffer: dissolve 7-10 g of ammonium acetate granules in 100 mL of deionized water and add acetic acid to adjust the pH to 4-5;

[0013] Prepare β-glucuronidase solution: dissolve 2-5 mg β-glucuronidase powder in 10 mL deionized water to obtain 250-500 U / mL β-glucuronidase solution;

[0014] Prepare the extraction solution: Mix 1-5 mL of urine with 1-5 mL of deionized water, and add 400-1000 μL of ammonium acetate buffer, 150-500 μL of β-glucuronidase solution, and 10-20 μL of a mixed solution containing 80 target compounds (concentration of 1 mg / L) in sequence;

[0015] After mixing, the solution was placed in a water bath at 36-40°C and incubated for 12 hours to obtain a solution to be extracted;

[0016] In the step 2), the aminated β-cyclodextrin composite material supported on cellulose is used as a solid phase extractant, and 2,3,5,6-tetrafluoroterephthalonitrile is used as a crosslinking agent and borane dimethyl sulfide is used for an amination reaction to prepare the composite material. The preparation yield of the composite material exceeds 50%, and the particle size of the composite material particles is 10 to 100 μm.

[0017] Furthermore, the extraction efficiency of endogenous and exogenous organic matter in urine samples remained consistent, and the recovery rate of target organic matter was highly consistent among different urine samples.

[0018] Furthermore, the average spike recoveries for urine ranged from 60% to 130%, and the relative standard deviations (RSDs) between different urines were less than 30%.

[0019] In the step 2), the amino-β-cyclodextrin composite material loaded on cellulose is used as a solid phase extraction agent to simultaneously adsorb endogenous and exogenous organic matter in urine without being interfered by the complex matrix in urine.

[0020] Furthermore, the method specifically comprises the following steps:

[0021] 1) Activation of the solid phase extraction column: Use methanol to activate the solid phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add deionized water for pre-equilibrium treatment;

[0022] 2) Sample loading: When the deionized water level is about to reach the upper sieve plate of the extraction column, add the solution to be extracted immediately, and keep the flow rate at 4-5mL / min to ensure that the sample passes through the extraction column evenly;

[0023] 3) Drying: After loading, air dry the extraction column to remove residual moisture;

[0024] 4) Elution: First add 3-5 mL of 0.5-1 M ammonium acetate methanol solution for preliminary elution. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 2-4 mL of 0.05-0.1 M CaCl2 methanol solution for the second step of elution, and collect all the eluent;

[0025] 5) Nitrogen blowing: Use a gentle nitrogen flow to blow the eluent to a completely dry state, and use the initial mobile phase of liquid chromatography to re-adjust the volume to 1-2 mL;

[0026] 6) Filtration: Filter the fixed volume solution through a filter membrane to remove particulate impurities and prevent clogging of the chromatographic column;

[0027] 7) The pretreated samples were injected into an ultra-high performance liquid chromatography-mass spectrometry system for analysis: First, a standard curve was established with the concentrations set at 0.1, 0.5, 1, 5, 10, and 50 μg / L. The recoveries of 80 target substances were calculated based on the standard curve. Three parallel experiments were set up and the relative standard deviation was calculated to evaluate the repeatability of the method.

[0028] Furthermore, in the liquid chromatography-mass spectrometry analysis step, UPLC-MS / MS is used to analyze the sample to be analyzed, a multiple reaction monitoring (MRM) mode is adopted, and a standard curve is established to calculate the recovery rate of the target organic matter.

[0029] Furthermore, the concentration of the solution to be extracted is 10-20 μg / L, and contains a mixture of at least 80 exogenous and endogenous organic substances.

[0030] The step 7) analyzing using a liquid chromatography-mass spectrometry system comprises the following steps:

[0031] 1) The present invention systematically optimizes the conditions of liquid chromatography, such as the chromatographic column, mobile phase, elution procedure and flow rate, to achieve efficient separation and detection of a variety of complex compounds.

[0032] The preferred chromatographic column is ZORBAX RRHD Bonus-RP (2.1×50 mm, 1.8 μm), and the column temperature is 40°C;

[0033] The preferred mobile phases are an aqueous solution containing 2 mM ammonium acetate (A) and a methanol solution containing 0.01% formic acid (B);

[0034] Preferably, the elution procedure is: 0-1min, the volume content of mobile phase A is maintained at 100%; 1-2.5min, the volume content of mobile phase A is adjusted from 100% to 50%, and the volume content of mobile phase B is adjusted from 0% to 50%; 2.5-4min, the volume content of mobile phase A is adjusted from 50% to 5% and maintained for 2min; 6-8min, the volume content of mobile phase A is adjusted from 5% to 100%, and the volume content of mobile phase B is adjusted to 0% accordingly;

[0035] Preferably, the flow rate is 0.5 mL / min;

[0036] 2) The present invention optimizes the mass spectrometry conditions to improve ionization efficiency and sensitivity and ensure the accuracy and stability of the detection results.

[0037] The preferred mass spectrometry system is a triple quadrupole linear ion trap mass spectrometer (SCIEX 6500+System, USA), equipped with electrospray ion source (ESI), multiple reaction monitoring (MRM) mode, positive and negative ion modes were collected simultaneously;

[0038] Preferred mass spectrometry conditions include: ion spray voltage of 5500 V and -4000 V, temperature of 550 ° C, nebulizer gas pressure of

[0039] The pressure was 50 psi, the auxiliary heating gas was 50 psi, the curtain gas was 40 psi, and the collision gas was 9 psi.

[0040] The present invention has the following beneficial effects: it is significantly superior to the existing technology in terms of recovery rate and accuracy, and is suitable for use in environmental monitoring, public health, disease diagnosis, biomedical research and other fields. At the same time, the present invention has a low analysis cost and a high sample processing efficiency, and provides a reliable technical means for large-scale sample analysis and commercial applications, which is specifically manifested as follows: 1. Efficient sample pretreatment and separation capabilities: The present invention provides an efficient solid phase extraction technology, which uses a cellulose-loaded amino-β-cyclodextrin composite material to significantly improve the extraction efficiency of 80 organic substances in urine (covering exogenous and endogenous substances). The composite material has excellent adsorption capacity and selectivity, can efficiently and simultaneously extract endogenous metabolites and exogenous pollutants in urine, and can effectively reduce the interference of the complex matrix of urine, and improve the recovery rate and detection sensitivity of the target compound.

[0041] 2. Optimization of high performance liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The present invention achieves efficient separation and sensitive detection of 80 target compounds by optimizing liquid chromatography and mass spectrometry conditions. In the liquid chromatography part, the chromatographic column, mobile phase, flow rate and gradient program are optimized, so that the complex endogenous and exogenous organic matter in the urine sample can be well separated in a short time, reducing the mutual interference between compounds. In the mass spectrometry part, the ionization efficiency and signal intensity of the target are significantly improved through the precise control of the ion source voltage, temperature and gas flow, ensuring high sensitivity and accuracy of the detection.

[0042] 3. High stability and wide application: The analytical method of the present invention has high accuracy and wide applicability, and is suitable for processing complex matrices such as urine. Compared with the commercially available HLB solid phase extraction column, the relative standard deviation (RSD) of the urine spike recovery rate obtained by the material of the present invention is significantly lower, indicating that the method has good accuracy and less matrix interference for different urine samples.

[0043] 4. The long-term storage conditions of the material have also been verified. When stored at room temperature, the material is not easy to degrade or fail, and the storage period can reach more than one year. In the performance test after more than one year of storage, the extraction efficiency did not change much compared with the fresh material, proving that it has good long-term stability.

[0044] 5. Cost analysis: The amino-β-cyclodextrin composite material loaded with cellulose has a significant cost advantage compared with the traditional solid phase extraction column (such as HLB solid phase extraction column). Cellulose, as a natural polymer material, has a low raw material cost and a wide range of sources, which can effectively reduce production costs. The synthesis method of the amino-β-cyclodextrin composite material is simple and the reaction conditions are mild, which can further reduce the raw material and production costs. Therefore, compared with the traditional HLB solid phase extraction column, the cost of the solid phase extraction column of the present invention can be reduced by at least 40%-50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The spiked recovery rates obtained by solid phase extraction of 80 organic substances in deionized water by the solid phase extraction method of the present invention in Example 3 are:

[0046] a is endogenous organic matter;

[0047] b and c are exogenous organic matter.

[0048] Figure 2 is the spiked recovery rate obtained by solid phase extraction of 80 organic substances in three actual urines by the solid phase extraction method of the present invention in Examples 4, 5 and 6:

[0049] a is endogenous organic matter;

[0050] b and c are exogenous organic matter.

[0051] Figure 3 The recoveries of 80 organic compounds in urine obtained by solid phase extraction using the solid phase extraction method of the present invention and the traditional solid phase extraction method are as follows:

[0052] a is endogenous organic matter;

[0053] b and c are exogenous organic matter. DETAILED DESCRIPTION

[0054] The present invention is further described below by specific examples and drawings. The embodiments of the present invention are intended to better enable those skilled in the art to better understand the present invention and do not impose any limitation on the present invention. The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art unless otherwise specified. The method for preparing a solid phase extractant of an amino-β-cyclodextrin composite material loaded on cellulose is the research result of the inventor, see CN116621266 A patent publication text for details.

[0055] Example 1

[0056] The specific steps of this embodiment include:

[0057] Step 1: Synthesis of β-cyclodextrin composites loaded on cellulose.

[0058] a) Add 6.0 g of β-cyclodextrin, 3.5 g of 2,3,5,6-tetrafluoroterephthalonitrile, 10.0 g of cellulose powder and 15.0 g of anhydrous potassium carbonate to 50 mL of dimethyl sulfoxide and mix well;

[0059] b) heating to 80° C. and stirring the reaction for 40 hours;

[0060] c) After cooling to room temperature, add 50 mL of tetrahydrofuran, stir and filter;

[0061] d) The filtered solid was added into a 250 mL conical flask, and 1 M HCl solution was added until no bubbles were generated;

[0062] e) After filtering, the mixture was washed with deionized water and methanol in sequence until neutral, and dried under vacuum at 60° C. for 10 hours to obtain a β-cyclodextrin composite material supported on cellulose.

[0063] Step 2: Synthesis of amino-β-cyclodextrin composite material supported on cellulose.

[0064] a) dispersing 10.0 g of the β-cyclodextrin composite material loaded on cellulose in 100 mL of tetrahydrofuran solution;

[0065] b) slowly adding 50 mL of borane dimethyl sulfide complex solution (concentration of 2 M, dissolved in tetrahydrofuran) in an ice bath, and stirring the reaction for 40 hours;

[0066] c) filtering the obtained solid after the reaction, and washing it with 1M HCl methanol solution, 5% NaOH aqueous solution, water and methanol in sequence until it becomes neutral;

[0067] d) drying the solid under vacuum at 60° C. for 10 hours to obtain an amino-containing β-cyclodextrin composite material supported on cellulose.

[0068] Example 2

[0069] The amino-β-cyclodextrin composite material loaded on cellulose prepared in Example 1 is used as a solid phase extraction agent to fill and assemble into a solid phase extraction column, which specifically includes the following steps:

[0070] S1 Select the extraction column: Take a clean extraction column with an effective capacity of 6 mL for use.

[0071] S2 bottom sieve plate installation: Before filling the extractant, a sieve plate is installed at the bottom of the extraction column to fix the filler and prevent the filler from flowing out of the column outlet.

[0072] S3 filling solid phase extraction agent: fill the extraction column with 0.2 g of amino-β-cyclodextrin composite material filler loaded on cellulose to ensure that the filler is evenly filled in the column.

[0073] S4 top sieve plate installation: Install another sieve plate on top of the filled filler to encapsulate the filler inside the extraction column.

[0074] S5 Packing Adjustment: Slightly adjust the packing to make it evenly spread on the bottom sieve plate, and make sure the upper sieve plate is parallel to the bottom sieve plate to ensure uniform flow of liquid during the extraction process.

[0075] Example 3

[0076] A solid phase extraction and instrumental analysis method for simultaneously analyzing 80 endogenous and exogenous organic compounds in deionized water, specifically comprising the following steps:

[0077] S1 Preparation of the solution to be extracted: a mixed solution containing 80 kinds of organic matter with a concentration of 10 μg / L was prepared with deionized water as the solution to be extracted.

[0078] S2 Activation of solid phase extraction column: Select the solid phase extraction column prepared in Example 2, add 5 mL of methanol in sequence, and when the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 5 mL of deionized water for pre-equilibrium treatment.

[0079] S3 loading: When the deionized water level is about to reach the upper sieve plate of the extraction column, immediately add 5 mL of the solution to be extracted and control the flow rate to maintain at 4-5 mL / min to ensure that the sample passes through the extraction column evenly.

[0080] S4 Drying: After loading, air dry the extraction column for 10 minutes to remove residual moisture.

[0081] S5 elution: first add 3-5 mL of 0.5-1 M ammonium acetate methanol solution for preliminary elution. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 2-4 mL of 0.05-0.1 M CaCl2 methanol solution for the second elution and collect all the eluate.

[0082] S6 Nitrogen blowing: Use a gentle nitrogen flow to blow the eluent to a completely dry state. Then, use the initial mobile phase of the liquid chromatography to re-adjust the volume to 1mL.

[0083] S7 Filtration: Filter the fixed volume solution through a 0.22 μm nylon filter membrane to remove particulate impurities and prevent clogging of the chromatographic column.

[0084] S8 Sample testing: The pretreated samples were injected into an ultra-high performance liquid chromatography-mass spectrometry system (UPLC-MS / MS) for analysis. First, a standard curve was established, and the concentrations were set to 0.1, 0.5, 1, 5, 10, and 50 μg / L. The recoveries of 80 target substances were calculated based on the standard curve, and three parallel experiments were set up and the relative standard deviation was calculated to evaluate the repeatability of the method. Figure 1 As shown in Figure 2, the recovery of endogenous organic matter in deionized water ranged from 60% to 118%, and the relative standard deviation of parallel experiments was within 10% ( Figure 1 a), showing the high stability and good repeatability of this method. Figure 1 c shows that among the exogenous pollutants, the recovery rates of 57 target substances are between 70% and 120%. Figure 1 b shows that only 7 exogenous pollutants had recoveries less than 50%, and the relative standard deviations (RSDs) of all exogenous pollutants were within 20%. The overall results showed that the method showed good accuracy and reliability in the detection of both endogenous and exogenous organic matter.

[0085] The chromatographic detection conditions of 80 endogenous and exogenous organic substances in this example are shown in Table 1.

[0086] Table 1

[0087]

[0088] The mass spectrometry detection conditions of this embodiment are as follows:

[0089] Instrument: Hybrid triple quadrupole linear ion trap mass spectrometer (SCIEX 6500+ System, USA), equipped with an electrospray ionization (ESI) source, and supporting both positive and negative ion modes.

[0090] Mode: Multiple reaction monitoring (MRM) mode.

[0091] Ion source voltage: set to 5500 V in positive ion mode and -4000 V in negative ion mode.

[0092] Gas settings: Curtain gas (CUR) at 40 psi, Nebulizer gas (GS1) and Auxiliary gas (GS2) at 50 psi.

[0093] Ion source temperature: 550°C.

[0094] In this example, basic information of 80 endogenous and exogenous organic substances is shown in Table 2.

[0095] Table 2

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In this example, 80 endogenous and exogenous organic ion pair parameters and standard curve correlation coefficients R 2 , LOD and LOQ are shown in Table 3.

[0102] Table 3

[0103]

[0104]

[0105]

[0106] Example 4

[0107] A solid phase extraction and instrumental analysis method for simultaneously analyzing 80 endogenous and exogenous organic compounds in urine using an amino-β-cyclodextrin composite material supported on cellulose as a solid phase extractant comprises the following steps:

[0108] S1 Urine Pretreatment:

[0109] Prepare ammonium acetate buffer: dissolve 7-10 g of ammonium acetate granules in 100 mL of deionized water and add acetic acid to adjust the pH to 4-5;

[0110] Prepare β-glucuronidase solution: dissolve 2-5 mg β-glucuronidase powder in 10 mL deionized water to obtain 250-500 U / mL β-glucuronidase solution;

[0111] Prepare the extraction solution: Mix 1-5 mL of urine with 1-5 mL of deionized water, and add 400-1000 μL of ammonium acetate buffer, 150-500 μL of β-glucuronidase solution, and 10-20 μL of a mixed solution containing 80 target compounds (concentration of 1 mg / L) in sequence;

[0112] After mixing, the solution was placed in a water bath at 36-40°C and incubated for 12 hours to obtain a solution to be extracted;

[0113] S2 Activation of solid phase extraction column: Select the solid phase extraction column prepared in Example 2, add 5 mL of methanol in sequence, and when the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 5 mL of deionized water for pre-equilibrium treatment.

[0114] S3 loading: When the deionized water level is about to reach the upper sieve plate of the extraction column, immediately add the solution to be extracted obtained in step S1, and control the flow rate to 4-5 mL / min to ensure that the sample passes through the extraction column evenly.

[0115] S4 Drying: After loading, air dry the extraction column for 10 minutes to remove residual moisture.

[0116] S5 elution: first add 3-5 mL of 0.5-1 M ammonium acetate methanol solution for preliminary elution. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 2-4 mL of 0.05-0.1 M CaCl2 methanol solution for the second elution and collect all the eluate.

[0117] S6 Nitrogen blowing: Use a gentle nitrogen flow to blow the eluent to a completely dry state. Then, use the initial mobile phase of the liquid chromatography to re-adjust the volume to 1mL.

[0118] S7 Filtration: Filter the fixed volume solution through a 0.22 μm nylon filter membrane to remove particulate impurities and avoid clogging of the chromatographic column.

[0119] S8 sample testing:

[0120] The pretreated samples were injected into an ultra-high performance liquid chromatography-mass spectrometry system (UPLC-MS / MS) for analysis. The recovery rates of 80 organic compounds were calculated based on the standard curve established in Example 1. If the recovery rate was within the range of 70% to 120%, the method had good accuracy.

[0121] Example 5

[0122] This example is a blank control of Example 4 and is used to calculate the recovery rate.

[0123] The difference between Example 5 and Example 4 is that in the urine pretreatment in S1, the mixed solution of 80 target compounds is not added, and the other steps are the same.

[0124] Example 6

[0125] The difference between Example 6 and Example 5 is that after nitrogen blowing in S6 and adjusting the volume to 1 mL, a mixed solution of 80 target substances is added to the sample to make the concentration of the target substance 10 μg / L, and the other steps are the same.

[0126] Examples 4, 5, and 6 all analyze urine from three groups of people of different age groups, specifically: infants (male, 1 year old); college students (female, 19 years old); and the elderly (female, 72 years old).

[0127] Recovery rate (%) = (Css-Cs) / (Cst-Cs)×100

[0128] Wherein, Css is the spiked sample concentration, i.e., the spiked sample measurement value obtained in Example 4; Cs is the unspiked sample concentration, i.e., the unspiked sample measurement value obtained in Example 5, also the urine matrix blank; Cst is the spiked concentration, i.e., the post-extraction spiked measurement value obtained in Example 6.

[0129] The recovery rates of the three urine samples were calculated based on the determination results of Examples 4, 5, and 6. The results are as follows: Figure 2 As shown in Figure 2, the average recovery rates of endogenous organic matter in the three urines ranged from 86% to 120%, and the relative standard deviations (RSDs) were all less than 20% ( Figure 2 a), showing the high efficiency and accuracy of the material of the present invention in extracting endogenous markers in urine. For exogenous pollutants, the average spike recovery of 58 substances in three different types of urine was between 80% and 120%, and the RSD was less than 20% ( Figure 2 c); the average recoveries of the eight substances were between 70% and 120%, but the RSDs were between 20% and 30%, indicating that there was a certain variability in the recoveries of these substances; in addition, the recoveries of seven substances were lower than 70% or higher than 120% ( Figure 2b), indicating that the extraction effects of these 7 substances are not ideal.

[0130] Comparative Example 1

[0131] This comparative experiment used the traditional HLB solid phase extraction column for pretreatment and detected 80 endogenous and exogenous organic substances in urine.

[0132] The difference between Comparative Example 1 and Example 4 is that an HLB solid phase extraction column with a specification of 6 mL and a filler of 200 mg is used for solid phase extraction.

[0133] Comparative Example 2

[0134] The difference between Comparative Example 2 and Example 5 is that an HLB solid phase extraction column with a specification of 6 mL and a filler of 200 mg is used for solid phase extraction.

[0135] Comparative Example 3

[0136] The difference between Comparative Example 3 and Example 6 is that an HLB solid phase extraction column with a specification of 6 mL and a filler of 200 mg is used for solid phase extraction.

[0137] The recovery rate was calculated in the same manner as in the example.

[0138] The average recovery rates of the embodiment and the comparative example were compared. The average recovery rate was calculated by the spiked recovery rates of three urine samples and the average value was taken. The relative standard deviation (RSD) was calculated to evaluate the accuracy of the two solid phase extraction materials. Figure 3 As shown. For endogenous markers, taking cortisol as an example, the recovery rate of the material of the present invention is significantly better than that of the commercially available HLB solid phase extraction column, and the RSD is smaller, showing higher accuracy ( Figure 3 a). For exogenous markers, HLB was more affected by matrix interference, which was reflected in: (1) the number of substances with recovery rates <70% or >120% was as high as 29, while there were only 7 in the materials of the present invention; (2) the number of target substances with RSD >20% was 25, which was significantly higher than the 8 in the materials of the present invention ( Figure 3 b,c).

[0139] In general, the material of the present invention shows significant superiority in the simultaneous detection of endogenous and exogenous substances in urine, overcoming the shortcomings of existing detection technologies, and especially solving the technical problem that existing methods need to handle endogenous and exogenous substances separately. This method achieves the simultaneous extraction and detection of 80 target substances in urine by optimizing sample pretreatment and instrument analysis processes, which is not only convenient and fast, but also effectively reduces the detection cost.

[0140] The specific results showed that: (1) the average recovery rates of the seven endogenous markers ranged from 80% to 120%, and the RSDs were all less than 20%; (2) among the 73 exogenous markers, the average recovery rates of 66 substances were between 70% and 120%, and only seven substances slightly exceeded this range; (3) compared with commercially available HLB solid phase extraction columns, the materials of the present invention showed significant advantages in terms of recovery rate, accuracy and resistance to matrix interference.

[0141] The present invention not only provides an efficient and economical solution for the simultaneous analysis of endogenous and exogenous substances in urine, but also provides important technical support for further exploring the impact of exposure to exogenous pollutants on human health and health exposure risk assessment, thereby broadening its application scope in environmental monitoring and public health research.

[0142] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Aminated β-cyclodextrin composite material loaded on cellulose was used as solid phase extraction agent for simultaneous analysis of endogenous and exogenous organic matter in urine.

2. A method for simultaneously analyzing endogenous and exogenous organic matter in urine using the amino-β-cyclodextrin composite material supported on cellulose as claimed in claim 1 as a solid phase extractant, characterized in that: The following steps are involved: 1) Urine pretreatment; 2) using an amino-β-cyclodextrin composite material supported on cellulose as a solid phase extractant; 3) Extracting urine using a solid phase extraction column; 4) performing liquid chromatography-mass spectrometry analysis on the extracted samples to detect endogenous and exogenous organic matter; The step 1) comprises: Prepare ammonium acetate buffer: dissolve 7-10g of ammonium acetate granules in deionized water and add acetic acid to adjust the pH to 4-5; Prepare β-glucuronidase solution: dissolve 2-5 mg β-glucuronidase powder in deionized water to obtain 250-500 U / mL β-glucuronidase solution; Prepare the extraction solution: Mix 1-5 mL of urine with 1-5 mL of deionized water, and add 400-1000 μL of ammonium acetate buffer, 150-500 μL of β-glucuronidase solution, and 10-20 μL of a mixed solution containing 80 target compounds in sequence; After mixing, the solution was placed in a water bath at 36-40° C. and incubated to obtain a solution to be extracted.

3. The solid phase extraction method according to claim 2, characterized in that: In the step 2), the aminated β-cyclodextrin composite material supported on cellulose is used as a solid phase extractant, and 2,3,5,6-tetrafluoroterephthalonitrile is used as a crosslinking agent and borane dimethyl sulfide is used for an amination reaction to prepare the composite material. The preparation yield of the composite material exceeds 50%, and the particle size of the composite material particles is 10 to 100 μm.

4. The method according to claim 2, characterized in that: The extraction efficiency of endogenous and exogenous organic matter in urine samples was consistent, and the recovery rate of target organic matter was highly consistent among different urine samples.

5. The method according to claim 2, characterized in that: The average spike recoveries for urine ranged from 60% to 130%, and the relative standard deviations between different urine samples were less than 30%.

6. The solid phase extraction method according to claim 2, characterized in that: In the step 2), the amino-β-cyclodextrin composite material loaded on cellulose is used as a solid phase extraction agent to simultaneously adsorb endogenous and exogenous organic matter in urine without being interfered by the complex matrix in urine.

7. The method according to claim 2, characterized in that The specific steps include: 1) Activation of the solid phase extraction column: Use methanol to activate the solid phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add deionized water for pre-equilibrium treatment; 2) Sample loading: When the deionized water level is about to reach the upper sieve plate of the extraction column, add the solution to be extracted immediately, and keep the flow rate at 4-5mL / min to ensure that the sample passes through the extraction column evenly; 3) Drying: After loading, air dry the extraction column to remove residual moisture; 4) Elution: First add 3-5 mL of 0.5-1 M ammonium acetate methanol solution for preliminary elution. When the methanol liquid level is about to reach the upper sieve plate of the solid phase extraction column, immediately add 2-4 mL of 0.05-0.1 M CaCl2 methanol solution for the second step of elution, and collect all the eluent; 5) Nitrogen blowing: Use a gentle nitrogen flow to blow the eluent to a completely dry state, and use the initial mobile phase of liquid chromatography to re-adjust the volume to 1-2 mL; 6) Filtration: Filter the fixed volume solution through a filter membrane to remove particulate impurities and prevent clogging of the chromatographic column; 7) Inject the pretreated samples into an ultra-high performance liquid chromatography-mass spectrometry system for analysis: First, establish a standard curve, calculate the recoveries of exogenous and endogenous organic matter based on the standard curve, set up three parallel experiments and calculate the relative standard deviation to evaluate the repeatability of the method.

8. The method according to claim 7, characterized in that The concentration of the solution to be extracted in step 2) is 10-20 μg / L, and contains a mixture of at least 80 exogenous and endogenous organic substances.

9. The method according to claim 7, characterized in that: In the step 7) of liquid chromatography-mass spectrometry analysis, UPLC-MS / MS is used to analyze the sample to be analyzed, a multiple reaction monitoring mode is adopted, and a standard curve is established to calculate the recovery rate of the target organic matter.

10. The method according to claim 7, characterized in that The step 7) analyzing using a liquid chromatography-mass spectrometry system comprises the following steps: 1) Optimization of liquid chromatography conditions: The mobile phases were an aqueous solution containing 2 mM ammonium acetate (A) and a methanol solution containing 0.01% formic acid (B); The elution procedure is as follows: 0-1min, the volume content of mobile phase A is maintained at 100%; 1-2.5min, the volume content of mobile phase A is adjusted from 100% to 50%, and the volume content of mobile phase B is adjusted from 0% to 50%; 2.5-4min, the volume content of mobile phase A is adjusted from 50% to 5% and maintained for 2min; 6-8min, the volume content of mobile phase A is adjusted from 5% to 100%, and the volume content of mobile phase B is adjusted to 0% accordingly; 2) Optimization of mass spectrometry conditions: The mass spectrometry conditions included: ion spray voltage of 5500 V and -4000 V, temperature of 550°C, nebulizer gas pressure of 50 psi, auxiliary heating gas of 50 psi, curtain gas of 40 psi, and collision gas of 9 psi.

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