Non-targeted screening method for organism drug metabolites based on solid-phase microextraction in-vivo sampling technology

Abstract

The invention discloses an organism drug metabolite non-targeted screening method based on a solid-phase microextraction in-vivo sampling technology, and relates to the technical field of organism drug metabolism research. The invention discloses a non-targeted screening method for organism drug metabolites. The non-targeted screening method comprises the following steps: extracting a target analyte of an organism under drug culture by utilizing a biocompatible solid-phase microextraction probe; soaking the extracted and sampled solid-phase microextraction probe in a desorption solvent, and desorbing the target analyte under oscillation to obtain a desorption solution; quantitatively adding a deuterated substance isotope internal standard into the desorption liquid; non-targeted screening is carried out on the target metabolite through liquid chromatography-tandem mass spectrometry, and the target metabolite is qualitatively and / or quantitatively analyzed. According to the method, the integrity of the biological sample is kept to the greatest extent. Meanwhile, continuous dynamic information of the drug metabolism process can be obtained, information of the drug metabolism process is accurately obtained, and a drug metabolism path is analyzed.

Description

Technical Field

[0001] The present invention relates to the technical field of biological drug metabolism mechanism research, and in particular to a method for non-targeted screening of biological drug metabolites based on solid phase microextraction in vivo sampling technology. Background Art

[0002] Various drugs remaining in the environment may be absorbed by organisms and undergo a series of metabolic processes. Their metabolites may not only have higher toxicity than the parent drug, interfering with the normal physiological activities of animals and plants, but also retain a certain degree of parent drug activity due to limited structural modifications, which may lead to unpredictable exposure risks to the human body through the food chain. Therefore, systematic analysis of drug biotransformation pathways and metabolites is of great scientific value for building a drug environmental risk warning system and formulating precise control strategies.

[0003] By using high-resolution mass spectrometry to perform non-targeted screening of drug metabolites in plants and animals, new drug metabolites can be discovered and identified, which is an important tool for analyzing the molecular mechanism of drug biotransformation. In the field of new metabolite screening, the commonly used sample pretreatment method is solvent extraction, that is, after a certain period of drug culture, a sufficient amount of biological tissue samples (usually 1-2g) are obtained through destructive separation (such as picking plant roots, stems, and leaves; separating muscle or organ tissues after anesthetizing animals to death, etc.), and then freeze-drying, grinding, solvent extraction, SPE enrichment and concentration are performed to obtain analytical samples. However, this method requires a large amount of destructive sampling of organisms, and it is impossible to achieve dynamic tracking of drug metabolism in the body in different regions. At the same time, complex pretreatment processes (such as freeze-drying, grinding, and multi-step extraction) may lead to the inactivation of unstable metabolites, resulting in the loss of information on key intermediates in the transformation path, which also increases the difficulty of implementing the research. Therefore, it is urgent to develop a new non-targeted screening method that is non-destructive, simple, and rapid to promote the study of drug metabolism mechanisms.

[0004] Solid phase microextraction (SPME) sampling technology is an efficient and sensitive sample pretreatment method. This technology integrates sampling, extraction, concentration and injection, and is easy to operate; it can also be used in conjunction with efficient separation and detection technologies such as chromatography and mass spectrometry, so it has the advantages of being easy to automate, easy to operate, not requiring solvents, and being environmentally friendly. However, the existing solid phase microextraction sampling technology is mainly aimed at sampling in vitro tissues. For example, patent CN115060816A is a headspace solid phase microextraction tandem gas chromatography-mass spectrometry detection method for fluamine metabolites in urine, which uses headspace solid phase microextraction technology (HS-SPME) to collect volatile substances of fluamine metabolites in urine. However, headspace solid phase microextraction technology requires heating or shaking, so headspace SPME sampling cannot be performed on larger living organisms such as animals and plants. There are no reports on the use of solid phase microextraction sampling technology for non-targeted screening of drug metabolites in vivo; nor are there any reports on the use of solid phase microextraction sampling technology for dynamic tracking of drug metabolism in vivo in different regions to conduct drug metabolism research. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of existing biological drug metabolite analysis and provide a non-targeted screening method for biological drug metabolites based on solid phase microextraction in vivo sampling technology, which can maintain the integrity of biological samples to the greatest extent, has high detection efficiency, and high accuracy of analysis results.

[0006] Another object of the present invention is to provide a method for studying the drug metabolism mechanism of an organism, which can achieve the capture and dynamic tracking of drug metabolites without interfering with the normal physiological state of the organism.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention protects a method for non-targeted screening of drug metabolites in an organism, comprising the following steps: S101, extracting target analytes from organisms cultured with drugs using a biocompatible solid phase microextraction probe; S102, washing the solid phase microextraction probe after extraction and sampling, immersing it in a desorption solvent, desorbing the target analyte under shaking, and obtaining a desorption solution; S103, quantitatively adding a deuterated isotope internal standard to the desorption solution to obtain a target analysis solution; S104, taking the target analysis liquid for detection by liquid chromatography-tandem mass spectrometry, screening out candidate metabolites based on the characteristics of the parent drug fragments and performing structural identification.

[0008] The present invention discloses a non-targeted screening method for drug metabolites in living organisms, which adopts a biocompatible SPME probe for in vivo sampling, simplifies the complex sample pretreatment process into a one-step process, and significantly improves the analysis efficiency. It breaks through the destructive sampling of traditional pretreatment methods and maintains the integrity of biological samples to the greatest extent. At the same time, the sampling conditions are mild, the sampling time is short, and no metabolic quenching treatment is involved, so that metabolites with less stable structures can be detected.

[0009] The organism of the present invention refers to an animal or plant with life characteristics. The present invention has no particular limitation on the species of the organism. Any organism applicable to the field of biological drug metabolism mechanism research can achieve the purpose of the present invention; illustratively, it includes but is not limited to murine animals and Asteraceae plants.

[0010] In some embodiments, step S101 specifically includes: using a hollow needle to make a wound at the target part of the organism under drug culture, placing a biocompatible solid phase microextraction probe into the inner cavity of the hollow needle, taking out the hollow needle to make the solid phase microextraction probe directly contact with the biological tissue, and extracting the target analyte. Through the minimally invasive sampling strategy, dynamic information of the drug metabolism process can be obtained without interfering with the normal physiological state of the organism.

[0011] In some embodiments, the wound depth is 1.5-2.5 cm.

[0012] In some embodiments, the outer diameter of the hollow needle is 200-300 μm, and the inner diameter thereof is larger than the outer diameter of the biocompatible solid phase microextraction probe.

[0013] Optionally, the sampling time of the solid phase microextraction probe is 15-30 min, preferably 18-22 min.

[0014] In some embodiments, in step S101, the surface coating material of the biocompatible solid phase microextraction probe is C18. Biocompatible materials are not toxic to animals and plants and are not easily rejected by organisms.

[0015] The preferred coating material of the present invention is C18. On the one hand, C18 has good biocompatibility and is not easily interfered by the accumulation of complex matrix macromolecules (sugar, protein) in the body; on the other hand, the C18 probe also has good broad spectrum. The modification process of biological exogenous substances involves complex mechanisms, and the properties of metabolites may undergo relatively large polarity changes. The C18 probe with good broad spectrum has relatively good extraction ability for non-polar to medium polar substances, which can meet the requirements of in vivo analysis. It is small in size, generally with a base of less than 200um and a coating thickness of 40-50um, and is not easy to interfere with the normal physiology of animals and plants.

[0016] In some embodiments, in step S101, a pre-equilibrium treatment of the solid phase microextraction probe is further included before sampling, and the pre-equilibrium treatment step includes: immersing the biocompatible solid phase microextraction probe in a desorption solvent and performing pre-equilibrium under shaking.

[0017] Solid phase microextraction is an equilibrium extraction technology used to measure the free concentration of analytes in samples, but it is also easily affected by conditions such as extraction time and vibration frequency. In order to improve its extraction performance, the present invention performs a pre-equilibrium treatment. The process parameters of the pre-equilibrium step and the desorption step of the present invention should be the same or substantially the same, and the process parameters include desorption solvent, treatment time and oscillation speed.

[0018] Optionally, in step S101 and step S102, the desorption solvent is a methanol / water solution in a volume ratio of (0.8-1.2):1; preferably a methanol / water solution in a volume ratio of 1:1 (v / v).

[0019] Optionally, the equilibration time is 25-40 min.

[0020] Optionally, the shaking is performed by a vortex shaker at a rotation speed of 450-550 rpm.

[0021] In some of the embodiments, in step S103, candidate metabolites are screened out based on the characteristics of the parent drug fragments and structural identification is performed, specifically including: based on the primary full-scan mass spectrometry data, candidate metabolites are screened based on the characteristics of the parent drug fragments, and then secondary mass spectrometry analysis is performed and compared with the literature / standard spectral library to confirm the structural identification.

[0022] Furthermore, if the organism is a plant, screening can be carried out in combination with the plant phase I metabolic rules, specifically including: screening candidate metabolites based on the primary full-scan mass spectrometry data, combined with the plant phase I metabolic rules and the characteristics of the parent drug fragments, and then performing secondary mass spectrometry analysis and comparison with the literature / standard spectral library to confirm the structure identification.

[0023] Phase I metabolism of plants is to increase the water solubility, polarity or reactivity of exogenous substances by making simple modifications to their molecular structures, so as to facilitate their transport in plants and conjugate with polar molecules. This simple modification reaction is mainly completed by catalysis of intracellular enzymes (such as cytochrome P450), including a series of reactions such as hydroxylation, alkylation, dealkylation, desaturation, decarboxylation, and dehydration. Since only simple modifications are made, the chemical structure of the metabolites is similar to that of the parent drug molecules, and some metabolites will show the same fragment ion characteristics in the secondary mass spectrometry, such as the core substructure of enrofloxacin is a fragment with a mass-to-charge ratio of 263.0826. Therefore, during the first-level full scan of non-targeted screening, the plant phase I metabolism rules and the parent drug fragment characteristics can help narrow the screening range and provide a directional reference for screening the parent ions of candidate metabolites, thereby improving the credibility of the metabolites obtained by screening.

[0024] The present invention protects a method for studying the drug metabolism mechanism of an organism, comprising the following steps: S201, extracting target analytes from an organism under drug culture at different time points using a biocompatible solid phase microextraction probe; S202, washing the biological tissue adhered to the solid phase microextraction probe after extraction and sampling, immersing the probe in a desorption solvent, desorbing the target analyte under shaking, and obtaining a desorption solution; S203, quantitatively adding a deuterated isotope internal standard to the desorption solution obtained at each time point to obtain a target analysis solution at different time points; S204, taking the target analysis solutions at different time points in step S203 in equal volumes, mixing and concentrating, and using liquid chromatography tandem mass spectrometry to screen candidate metabolites based on the characteristics of parent drug fragments and perform structural identification; S205, respectively taking the target analysis liquid at different time points in step S203, and analyzing the identified target metabolites by liquid chromatography-tandem mass spectrometry; normalizing the target metabolite data at different time points with the deuterated isotope internal standard as a reference, and determining the temporal changes in the drug metabolism pathway and the metabolite content.

[0025] Preferably, the different time points are sampling time points at fixed time intervals, and the time intervals may be 6h, 12h, 24h or 36h; illustratively, the different time points are specifically sampling at 0 o'clock on the 1st, 3rd, 5th, 7th, 10th and 14th days after administration.

[0026] The present invention provides a method for studying the drug metabolism mechanism of an organism, which can obtain continuous dynamic information of the drug metabolism process without interfering with the normal physiological state of the organism, and overcome the defects of the traditional pre-treatment method in destructive sampling, metabolite loss and time dimension information loss. At the same time, its mild sampling conditions effectively protect the integrity of unstable metabolic intermediates, ensure the accurate acquisition of drug metabolism process information, and accurately analyze the drug metabolism pathway.

[0027] In some embodiments, in step S204, candidate metabolites are screened and structural identification is performed, specifically including: based on the primary full-scan mass spectrometry data, the candidate metabolites are screened in combination with the plant phase I metabolism rules and the parent drug fragment characteristics, and then the secondary mass spectrometry analysis is performed and compared with the literature / standard spectral library to confirm the structural identification.

[0028] Optionally, liquid chromatography conditions: chromatographic separation is performed using a C18 column (Thermo Scientific Hypersil GOLD column, 2.1 mm × 100 mm id, 1.9 μm), column temperature is 35°C. The mobile phase is 0.1% formic acid aqueous solution (A) and methanol (B), and the flow rate is 0.20 mL min -1 , separation gradient: 0-1 min, 90 % A-90 % A; 1-25 min, 90 % A-3 % A; 25-30 min, 3 % A-3 % A; 30-39 min, 3 % A-90 % A; 39 min-45 min, 90 % A-90 % A. The injection volume was 5 μL.

[0029] Optionally, the mass spectrometry conditions are as follows: mass spectrometry analysis is performed in positive ion mode, the spray voltage is 3500 V, the sheath gas and auxiliary gas flow rates are 30 arb and 10 arb, respectively, and the ion transfer tube and evaporator temperatures are 320°C and 350°C, respectively. Full scan (MS1) is performed using an Orbitrap detector with a resolution of 70000, a mass range of 50-750Da, and a maximum injection time of 200 ms. The candidate biotransformation products obtained from the full scan screening are scanned by secondary mass spectrometry (MS2) with a resolution of 17500, a maximum injection time of 100 ms, and HCD step collision energies set to 20, 30, and 50.

[0030] In some embodiments, the drug includes enrofloxacin. Enrofloxacin metabolites are of various types, unknown structures, and low concentrations in biological samples. The chemical properties of the metabolites are similar, making analysis difficult. The present invention uses SPME probes for in vivo sampling, combined with liquid chromatography-high-resolution mass spectrometry technology, to accurately identify and analyze enrofloxacin metabolites, and can provide technical support for the study of its drug biological metabolism mechanism.

[0031] In some embodiments, the deuterated isotope internal standard is enrofloxacin-d5.

[0032] Preferably, the final concentration of enrofloxacin-d5 is 15-25 μg / L.

[0033] In some embodiments, the drug metabolite comprises at least one of the following compounds of formula I to formula IV: (I) (Ⅱ) (III) (IV).

[0034] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for non-targeted screening of drug metabolites in an organism, which adopts a biocompatible SPME probe for in vivo sampling to achieve non-targeted screening of drug metabolites, with high analysis efficiency and accurate analysis results.

[0035] The SPME probe sampling simplifies the complex sample pretreatment process into a one-step process, significantly improving the analysis efficiency; it breaks through the destructive sampling of traditional pretreatment methods and maintains the integrity of biological samples to the greatest extent.

[0036] The present invention provides a method for studying the drug metabolism mechanism of an organism, which can obtain continuous dynamic information of the drug metabolism process without interfering with the normal physiological state of the organism, and overcome the defects of the traditional pre-treatment method in destructive sampling, metabolite loss and missing information in the time dimension. At the same time, its sampling conditions are mild, time-consuming, and the integrity of unstable metabolic intermediates is protected. It can accurately obtain instantaneous metabolites, ensure the accuracy and comprehensiveness of the information obtained on the drug metabolism process, and accurately analyze the drug metabolism pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The secondary mass spectrum of the enrofloxacin metabolites in Example 1 of the present invention is shown in FIG. The red line represents the fragment ion peak that matches the standard library (matching ≥2 characteristic fragment ions, mass deviation <5 ppm).

[0038] Figure 2 This is a comparison chart of the concentrations of enrofloxacin and ciprofloxacin determined by the methods of Example 1 of the present invention and Comparative Example 1. Wherein, ns means no significant difference.

[0039] Figure 3 This is a trend chart of the relative content of the enrofloxacin metabolites identified in Example 2 of the present invention, wherein ND means not detected. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0041] Example 1 A non-targeted screening method for plant living antibiotic metabolites based on solid phase microextraction living sampling technology comprises the following steps: S1. Pre-equilibration: Place the C18 coated SPME probe in a methanol / water solution (1:1, v / v) and vortex at 500 rpm for 30 min.

[0042] S2. SPME probe sampling: At fixed time points on the 1st, 3rd, 5th, 7th, 10th, and 14th days after the lettuce plants were administered with enrofloxacin (5 mg / L), a 2 cm deep micro-incision was made on the lettuce stem using a hollow needle (150 um inner diameter). The SPME probe was carefully pushed into the needle, and then the needle was removed so that its C18 coating was in contact with the plant tissue for 20 minutes to complete the live sampling. S3, desorption: After sampling, the SPME probe was removed, and the probe surface was gently rinsed with deionized water to remove possible adhered biological tissues, and then blotted with dust-free paper, and then immersed in a methanol-water solution (1:1, v / v) and vortexed for desorption at 500 rpm for 30 min. Then, enrofloxacin-d5 internal standard solution (final concentration 20 μg / L) was added to the desorption solution for instrument response correction and mass spectrometry data peak alignment to ensure the accuracy and reproducibility of the analysis results; S4. Non-targeted screening: Take 20 μL of the desorption solution at each time point and mix them in equal amounts. After blowing them nearly dry with nitrogen, re-dissolve them with 100 μL of methanol and analyze them using ultra-high performance liquid chromatography-tandem orbitrap high-resolution mass spectrometry. The specific analysis conditions are as follows: (1) Chromatographic conditions: A Thermo Scientific Hypersil GOLD column (2.1 mm × 100 mm id, 1.9 μm) was used for chromatographic separation. The column temperature was maintained at 35°C. The mobile phase was 0.1% formic acid in water (A) and methanol (B). The flow rate was 0.20 mL min -1 , separation gradient: 0-1 min, 90 % A-90 % A; 1-25 min, 90 % A-3 % A; 25-30 min, 3 % A-3 % A; 30-39 min, 3 % A-90 % A; 39 min-45 min, 90 % A-90 % A. The injection volume was 5 mL.

[0043] (2) Mass spectrometry conditions: Mass spectrometry analysis was performed in positive ion mode, with a spray voltage of 3500 V, sheath gas and auxiliary gas flow rates of 30 arb and 10 arb, respectively, and ion transfer tube and evaporator temperatures of 320°C and 350°C, respectively. Full scan (MS1) was performed using an Orbitrap detector with a resolution of 70,000, a mass range of 50 to 750 Da, and a maximum injection time of 200 ms. The candidate biotransformation products screened by MS1 were scanned by secondary mass spectrometry (MS2) with a resolution of 17,500, a maximum injection time of 100 ms, and HCD step collision energies set to 20, 30, and 50.

[0044] Based on the primary full-scan mass spectrometry data, candidate metabolites were screened in combination with the plant phase I metabolic rules and the characteristics of the parent drug fragments; then, the identification was completed through secondary mass spectrometry analysis and comparison with the literature / standard spectral library (matching ≥2 characteristic fragment ions, mass deviation <5 ppm).

[0045] The results are as follows Figure 1 As shown, four enrofloxacin metabolites, including ENRO-M1 to ENRO-M4, were successfully identified in lettuce using this method. The method of the present invention has the advantages of simple operation, high sensitivity, low organic solvent consumption, and accurate results.

[0046] Comparative Example 1 A non-targeted screening method for plant living antibiotic metabolites, which is different from Example 1 in that: This comparative example adopts solvent extraction method for sampling and processing, and the specific steps are as follows: (1) In vivo administration of enrofloxacin to lettuce plants On the 14th day after in vivo administration of enrofloxacin (5 mg / L), the lettuce plants were killed and the stem segments were collected, freeze-dried with liquid nitrogen and ground into powder.

[0047] (2) Weigh 0.5 g of powder and homogenize in a glass homogenizer containing 5 mL of methanol in an ice bath to form a homogenate. Place the homogenate in an ice bath and perform ultrasonic extraction for 30 min, then centrifuge and retain the supernatant and precipitate. (3) Place the precipitate in a glass homogenizer containing 5 mL of acetonitrile and homogenize it in an ice bath to form a homogenate. Place the homogenate in an ice bath and perform ultrasonic extraction for 30 min, then centrifuge and retain the supernatant and precipitate. (4) Combine the supernatants from (2) and (3), blow dry in a 40 °C water bath under nitrogen, redissolve in 1 mL of methanol, and dilute with 10 mL of deionized water; (5) Use a solid phase extraction (SPE) cartridge for purification. Add 6 mL of methanol and 6 mL of deionized water to the SPE cartridge for pretreatment. (6) Slowly add the diluted sample to the SPE column and then elute with 7 mL of methanol; (7) The eluate was dried with nitrogen in a 40 °C water bath, re-dissolved in 0.2 mL of methanol, filtered through a 0.22 μm filter membrane, and stored in a 4 °C refrigerator. The assay was completed within two weeks.

[0048] The non-targeted screening step is the same as step S104 of Example 1.

[0049] like Figure 2 As shown, the concentrations of enrofloxacin and ciprofloxacin determined by the solvent extraction method of Comparative Example 1 and the in vivo SPME extraction method of Example 1 were compared; the comparative analysis based on enrofloxacin (ENRO) and its representative metabolite ciprofloxacin (CIP) showed that there was no significant difference in the detection results between the solid phase microextraction in vivo sampling method and the conventional solvent extraction method (p>0.05), verifying the reliability of the method of the present invention.

[0050] Example 2 A method for studying the metabolomics of biological living drugs comprises the following steps: 1. The sample was extracted by the method of Example 1, and the desorption liquid at each time point obtained in step S3 was analyzed by ultra-high performance liquid chromatography-tandem orbitrap high-resolution mass spectrometry to simultaneously detect enrofloxacin-d5 and the enrofloxacin metabolites identified in Example 1; 2. Based on the peak area of ​​enrofloxacin-d5, the peak area of ​​the target metabolites was normalized to determine the drug metabolism pathway and analyze the temporal changes of metabolites.

[0051] The results are as follows Figure 3 As shown, the present invention dynamically monitored the metabolites of enrofloxacin in lettuce for 14 days, accurately tracked the changes in the relative contents of four drug metabolites from ENRO-M1 to ENRO-M4, and revealed the temporal changes in their relative contents. At the same time, the time dimension information provided by this method can accurately capture the generation characteristics of transient metabolites such as ENRO-M3, and through the temporal change analysis of metabolites, it can provide key data support for the study of drug biological metabolism mechanism.

[0052] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for non-targeted screening of drug metabolites in a biological body, characterized in that: The following steps are involved: S101, extracting target analytes from organisms cultured with drugs using a biocompatible solid phase microextraction probe; S102, washing the solid phase microextraction probe after extraction and sampling, immersing it in a desorption solvent, desorbing the target analyte under shaking, and obtaining a desorption solution; S103, quantitatively adding a deuterated isotope internal standard to the desorption solution to obtain a target analysis solution; S104, taking the target analysis liquid for detection by liquid chromatography-tandem mass spectrometry, screening out candidate metabolites based on the characteristics of the parent drug fragments and performing structural identification.

2. The method for non-targeted screening of biological drug metabolites according to claim 1, characterized in that: In step S101, specifically: a hollow needle is used to make a wound at the target site of the organism under drug culture, a biocompatible solid phase microextraction probe is placed into the inner cavity of the hollow needle, and the hollow needle is removed to make the solid phase microextraction probe directly contact with the biological tissue to extract the target analyte.

3. The method for non-targeted screening of biological drug metabolites according to claim 2, characterized in that: The wound depth is 1.5-2.5 cm.

4. The method for non-targeted screening of biological drug metabolites according to claim 1 or 2, characterized in that: In step S101, the surface coating material of the biocompatible solid phase microextraction probe is C18.

5. The method for non-targeted screening of biological drug metabolites according to claim 1, characterized in that: In step S101, the solid phase microextraction probe is pre-balanced before sampling. The pre-balanced step includes: immersing the biocompatible solid phase microextraction probe in a desorption solvent and performing pre-balance under shaking.

6. The method for non-targeted screening of biological drug metabolites according to claim 1 or 5, characterized in that: The desorption solvent is a methanol / water solution with a volume ratio of (0.8-1.2):

1.

7. A method for studying the drug metabolism mechanism of an organism, characterized in that: The following steps are involved: S201, extracting target analytes from an organism under drug culture at different time points using a biocompatible solid phase microextraction probe; S202, washing the biological tissue adhered to the solid phase microextraction probe after extraction and sampling, immersing the probe in a desorption solvent, desorbing the target analyte under shaking, and obtaining a desorption solution; S203, quantitatively adding a deuterated isotope internal standard to the desorption solution obtained at each time point to obtain a target analysis solution at different time points; S204, taking the target analysis solutions at different time points in step S203 in equal volumes, mixing and concentrating, and using liquid chromatography tandem mass spectrometry to screen candidate metabolites based on the characteristics of parent drug fragments and perform structural identification; S205, respectively taking the target analysis liquid at different time points in step S203, and analyzing the identified target metabolites by liquid chromatography-tandem mass spectrometry; normalizing the target metabolite data at different time points with the deuterated isotope internal standard as a reference, and determining the temporal changes in the drug metabolism pathway and the metabolite content.

8. The method for studying the drug metabolism mechanism of a living organism according to claim 7, characterized in that: In step S204, candidate metabolites are screened and their structures are identified, specifically including: based on the primary full scan mass spectrometry data, candidate metabolites are screened based on the characteristics of the parent drug fragments, and then secondary mass spectrometry analysis is performed to confirm the structure by comparing with the literature / standard spectral library.

9. The method for studying the drug metabolism mechanism of a living organism according to claim 7, characterized in that: The drug includes enrofloxacin.

10. The method for studying the drug metabolism mechanism of a living organism according to claim 9, characterized in that: Drug metabolites include at least one of the following compounds: （Ⅰ）、 （Ⅱ）、 （Ⅲ）、 （Ⅳ）。

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