Simple method, system and kit for characterizing CYP2E1 activity

Abstract

Traditional CYP2E1 activity detection adopts an elimination phase 6-hydroxy chlorzoxazone and chlorzoxazone ratio, but is interfered by UGT enzyme elimination, so that activity evaluation is distorted, and the CYP2E1 metabolism generation capability cannot be truly reflected. The invention discloses a simple method, system and kit for characterizing CYP2E1 activity. The method comprises the following steps: a, obtaining a biological sample after chlorzoxazone is administered; b, determining the concentration ratio of 6-hydroxy chlorzoxazone to chlorzoxazone of the sample at the highest time point of the absorption phase; the highest time point of the absorption phase is the peak reaching time of the concentration of 6-hydroxychlorzoxazone. It is found that after chlorzoxazone is applied to an organism, blood should be taken from the absorption phase of chlorzoxazone, and the activity of CYP2E1 can be really reflected.

Description

Technical Field

[0001] This invention belongs to the field of drug metabolism technology, specifically relating to the characterization of CYP2E1 activity. Background Technology

[0002] Cytochrome P450 2E1 (CYP2E1) is an important member of the CYP450 superfamily, mainly located in the endoplasmic reticulum and mitochondria of the liver, accounting for approximately 24.8% of total CYP450 enzymes. It plays a crucial role in regulating metabolism and inflammation. CYP2E1 not only participates in the metabolism of various endogenous substances such as arachidonic acid, linoleic acid, linolenic acid, and steroids, but also in the transformation of various exogenous drugs and procarcinogens such as ethanol, acetone, toluene, chloroform, carbon tetrachloride, and nitrosamines. Reports indicate that CYP2E1 is associated with various diseases, such as diabetes, obesity, gastric cancer, colorectal cancer, lung cancer, and bladder cancer. Therefore, exploring how to characterize CYP2E1 activity is of great significance.

[0003] Currently, the in vivo activity of CYP2E1 is determined by the rate at which the CYP2E1 metabolic probe drug chlorzoxazone is converted to 6-hydroxychlorzoxazone. This is typically achieved by collecting blood multiple times (6-10 times) after chlorzoxazone administration and characterizing the activity using pharmacokinetic parameters. However, due to the numerous problems associated with multiple blood collections, efforts have been made for many years to explore simpler methods, such as the single-point method (one blood collection), for determining CYP2E1 activity. Currently, the ratio of elimination-phase chlorzoxazone to its metabolite 6-hydroxychlorzoxazone is often used to characterize CYP2E1 activity. However, the concentration of elimination-phase 6-hydroxychlorzoxazone in blood is primarily affected by its elimination, reflecting the activity of its metabolic enzyme glucuronyltransferase, rather than the activity of the chlorzoxazone metabolic enzyme CYP2E1. Summary of the Invention

[0004] According to one aspect of this application, a simplified method for characterizing CYP2E1 activity is provided. Compared with the complex methods of the prior art, this simplified method does not require 6-10 samplings to obtain pharmacokinetic parameters for characterization, but instead obtains them in one step using specific indicators and algorithms of this invention. The simplified method includes the following steps: a. Obtain biological samples after administration of chlorzoxazone; b. Determine the concentration ratio of 6-hydroxychlorozoxazone to chlorzoxazone in the sample at the highest time point of the absorption phase; the highest time point of the absorption phase is the time when the concentration of 6-hydroxychlorozoxazone reaches its peak.

[0005] This invention discovers that after administration of chlorzoxazone, blood should be drawn during the absorption phase of chlorzoxazone (i.e., the formation phase of 6-hydroxychlorozoxazone) to truly reflect CYP2E1 activity. In studying mouse models with different trends in CYP2E1 activity, it was found that the elimination rate of 6-hydroxychlorozoxazone changed significantly in both the high CYP2E1 activity model induced by isoniazid and the low CYP2E1 activity model treated with carbon tetrachloride. However, the effect of 6-hydroxychlorozoxazone elimination during the absorption phase on its formation did not differ significantly between groups, indicating that blood drawn during the absorption phase, rather than the elimination phase, is more representative of CYP2E1 activity. This invention uses mouse administration of chlorzoxazone as an example for illustration.

[0006] Optionally, the biological sample is plasma or serum.

[0007] Optionally, the peak absorption time point is 5-20 minutes after administration in mice; 5-30 minutes in rats; and 0.5-2 hours in humans.

[0008] More preferably, the peak absorption time point is 10-20 minutes after administration to mice, 10-20 minutes to rats, and 1-2 hours to humans.

[0009] More preferably, the upper and lower limits of the highest absorption time point are independently selected from any point among 5-20 minutes after drug administration in mice, 5-30 minutes in rats, and 0.5-2 hours in humans.

[0010] Optionally, the administration method in step a is intravenous or gavage; the dose of chlorzoxazone is 10-100 mg / kg.

[0011] Preferably, the dose of chlorzoxazone administered in step a is 50-70 mg / kg for mice; 20-40 mg / kg for rats; and 5-15 mg / kg for humans. More preferably, the dose of chlorzoxazone administered in step a is 60 mg / kg for mice, 30 mg / kg for rats, and 10 mg / kg for humans.

[0012] Optionally, the administration method in step a is intravenous or gavage; the upper and lower limits of the chlorzoxazone dose are independently selected from any one or any two of the following: 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, and 100 mg / kg.

[0013] In this application, the concentration ratio is calculated using the following formula: .

[0014] Optionally, step a is performed in a model applicable to changes in CYP2E1 metabolic activity (the changes in activity include upregulation, downregulation, and no change). Optionally, the biological sample is obtained through the following processing steps: Incubate with β-glucuronidase; After extraction with ethyl acetate, the sample was dried under nitrogen. The mobile phase was reconstituted and the concentration was detected by LC-MS / MS.

[0015] According to another aspect of this application, a CYP2E1 metabolic activity detection system is provided, comprising: The sample processing module is used to perform the following biological sample processing steps: incubation with β-glucuronidase; extraction with ethyl acetate followed by drying with nitrogen; reconstitution with the mobile phase and detection of concentration by LC-MS / MS.

[0016] The ratio calculation module is used to calculate and output the CYP2E1 metabolic activity based on the detected concentration according to the following formula;

[0017] According to another aspect of this application, a kit for detecting CYP2E1 metabolic activity is provided, comprising: chlorzoxazone preparation, sample processing kit, and detection kit; Chlorzoxazone preparations: injectable solutions with concentrations of 1-100 mg / mL; Sample processing kit: β-glucuronidase, ethyl acetate, and reconstituted mobile phase; Test kit: LC-MS / MS standard for 6-hydroxychlorozoxazone and chlorozoxazone.

[0018] The beneficial effects that this application can produce include: I. Improved technical accuracy This invention utilizes the highest absorption time point (t) max The ratio of 6-hydroxychlorozoxazone to chlorzoxazone concentrations characterizes CYP2E1 activity and significantly improves detection accuracy. Specific results are as follows: 1. Solving the problem of phase interference in existing technologies: Traditional methods use elimination phase ratios (e.g., 45-60 minutes after administration), which are affected by the activity of the metabolic enzyme UGT. Changes in the elimination rate of 6-hydroxychlorozoxazone lead to inaccurate activity assessment. The method of this invention collects blood during the absorption phase (ascent phase) and uses t... max Using time points (e.g., 5-20 minutes in mouse models) avoids eliminating interference and directly reflects the production activity of CYP2E1. Experiments have shown that the absorption phase t maxThe ratios showed high correlation with microparticle activity, with isoniazid and carbon tetrachloride models showing r=0.57, p<0.007, r>0.83, and p<0.001, respectively. This fully demonstrates the detection advantage of this invention in eliminating phase interference.

[0019] 2. Highly consistent with in vitro microsomal activity evaluation methods: In models induced by isoniazid and treated with carbon tetrachloride, the ratio of the highest point of the absorption phase was significantly correlated with liver microsomal activity (which simply reflects the metabolic activity of the enzyme) (r=0.57, r=0.83), and the correlation was superior to other methods. This ensures the reliability of the data and provides a precise benchmark for drug metabolism research. The accuracy of the technique was verified through correlation analysis.

[0020] II. Ease of Operation and Efficiency This invention simplifies the CYP2E1 activity detection process and significantly improves experimental efficiency: 1. Single-point blood sampling reduces complexity: Traditional methods require multiple blood samplings (6-10 times) to construct a complete pharmacokinetic curve for calculating parameters such as AUC or CL. This method only requires sampling at the peak absorption phase (t... max With a single blood sample, activity can be characterized using a simple ratio formula. This reduces the difficulty of experimental procedures, animal / sample consumption (e.g., reducing the number of blood samples by 80% in mouse models), and significantly shortens the detection time to within 30 minutes.

[0021] 2. Standardized and easy-to-implement procedures: The method is equipped with clearly defined sample processing steps (such as β-glucuronidase incubation + ethyl acetate extraction + LC-MS / MS detection). In specific implementations, the procedure has shown good reproducibility in various models (such as gene knockout mice), ensuring rapid deployment.

[0022] III. The model has good universality. This invention demonstrates universality across diverse CYP2E1 activity variation models, ensuring broad applicability: 1. Coverage of bidirectional activity change models: The method has been effectively validated in both upregulation (e.g., isoniazid-induced) and downregulation (e.g., CYP2E1 gene knockout, Q11 inhibitor treatment, carbon tetrachloride liver injury) models. For example, in the isoniazid and carbon tetrachloride models, t max The ratio was significantly correlated with the activity.

[0023] 2. Cross-species compatibility: The method's principles are applicable to mammalian samples (such as mice, rats, and humans) because the absorption phase metabolic kinetics are similar (e.g., rat t...). max For 5-30 minutes, people t max (0.5-2 hours).

[0024] IV. Practical Application Value This invention provides an efficient tool for drug development and basic research, with specific applications including: 1. Accelerate drug development: By simplifying activity assays, this method supports high-throughput screening of CYP2E1 inhibitors or inducers (e.g., Q11 inhibitor validation) and predicts drug-drug interaction risks. For example, in the development of antidiabetic drugs, this method can rapidly assess the inhibitory strength of candidate compounds against CYP2E1.

[0025] 2. Advancing Disease Mechanism Research: CYP2E1 activity is associated with metabolic diseases (such as diabetes and obesity) and cancer. This method provides reliable activity data to help investigate the mechanism of action of CYP2E1 in pathological models (such as carbon tetrachloride-induced liver fibrosis).

[0026] 3. Reduced costs and resource consumption: Compared with traditional multiple blood collection methods, this approach reduces the number of experimental animals used (in the specific implementation, each group of mice can be reduced to 5-6 to complete the testing and verification) and reduces testing costs (LC-MS / MS single analysis is sufficient). Attached Figure Description

[0027] Figure 1 This document presents the pharmacokinetic curves of chlorzoxazone and 6-hydroxychlorzoxazone in mice, rats, and humans treated with isoniazid, Q11, cyp2e1 knockout, and carbon tetrachloride, according to one embodiment of this application. Figure 1 A and B are the drug-time curves of chlorzoxazone and 6-hydroxychlorzoxazone in mice treated with isoniazid. Figure 1 C and D are the drug-time curves of chlorzoxazone and 6-hydroxychlorzoxazone in Q11 and cyp2e1 knockout mice. Figure 1 E and F are the drug-time curves of chlorzoxazone and 6-hydroxychlorzoxazone in mice treated with carbon tetrachloride. Figure 1 G and H are the 6-hydroxychlorozoxazone time curves after oral administration of chlorzoxazone to rats and the 6-hydroxychlorozoxazone time curves after oral administration of chlorzoxazone to humans, respectively. Figure 2 This application describes the sensitivity of pharmacokinetic parameters AUC, CL, and t1 / 2 in different models to characterize changes in CYPE1 activity in one embodiment of the present application. Figure 2 Figure AC represents the isoniazid model, and Figure DF represents the Q11 and cyp2e1 knockout models. Figure 2 GI is the carbon tetrachloride model; Figure 3 This invention provides an inter-group comparison of the changes in the elimination rate of 6-hydroxychloroxazoline in the elimination phase and the effect of elimination on formation in the absorption phase in a carbon tetrachloride model of isoniazid and carbon tetrachloride in one embodiment of the present application. Figure 3A and B represent the elimination rates of 6-hydroxychlorozoxazone; Figure 3 The effect of elimination of 6-hydroxychlorozoxazone in the C and D absorbent phases on the production was compared between groups. Figure 4 To illustrate the ratio of 6-hydroxychlorozoxazone to chlorzoxazone and microsomal activity at different time points in one embodiment of this application, t 1 / 2 Correlation; among which Figure 4 AC represents the correlation between the ratio of 6-hydroxychlorozoxazone to chlorzoxazone and microsomal activity, while DF represents the correlation between the ratio of 6-hydroxychlorozoxazone to chlorzoxazone and t1 / 2. Detailed Implementation

[0028] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0029] Unless otherwise specified, all materials in the embodiments of this application were purchased commercially, and the CYP2E1 specific inhibitor Q11 was synthesized in our laboratory.

[0030]

[0031] The analysis method in the embodiments of this application is as follows: LC-MS / MS analysis was performed using an AB SCIEX 4500 instrument.

[0032] The specific testing conditions are as follows: Mass spectrometry and liquid phase conditions

[0033] Mass spectrometry conditions 2

[0034] Washing process

[0035] According to one embodiment of this application, the steps include: Step 101: Obtain biological samples after administration of chlorzoxazone; Step 102: Determine the concentration ratio of 6-hydroxychlorozoxazone to chlorzoxazone in the sample at the highest time point of the absorption phase; the highest time point of the absorption phase is the time when the concentration of 6-hydroxychlorozoxazone reaches its peak.

[0036]

[0037] Example 1 In this embodiment, the reagent package used is: Chlorzoxazone preparations: injectable solutions with concentrations of 1-100 mg / mL; Sample processing kit: β-glucuronidase, ethyl acetate, and reconstituted mobile phase; Test kit: LC-MS / MS standard for 6-hydroxychlorozoxazone and chlorozoxazone.

[0038] The specific implementation steps are as follows: 1. Preparation of mouse liver microsomes Solution preparation: 0.1 M Tris-Cl (pH 7.4): Weigh 1.21 g of Tris, dissolve in about 80 mL of distilled water, adjust the pH to 7.4 with concentrated hydrochloric acid, and add water to bring the volume to 100 mL. Store at 4℃ for later use; 3 mM Chlorzoxazone (CZX) solution: Weigh 50.70 mg of CZX, add 8 mL of ultrapure water and an appropriate amount of 1M NaOH solution, dissolve CZX completely, and add ultrapure water to bring the volume to 10 mL to obtain a 30 mM CZX stock solution. Dilute the stock solution to obtain a 3 mM CZX solution; NADPH: Weigh 25.00 mg of NADPH, dissolve in 3 mL of ultrapure water, dispense into aliquots, and store at -30℃. Liver microsomes were prepared using differential centrifugation. Liver specimens were thawed and weighed. A 1:4 (w / v) ratio of 50 mM Tris-HCl (pH=7.0) buffer (containing 150 mM KCl and 2 mM EDTA) was added, and the mixture was homogenized using a glass homogenizer to prepare a liver homogenate. The homogenate was centrifuged at 9000×g for 20 min at 4 °C, and the supernatant was centrifuged at 100000×g for 60 min at 4 °C. The resulting precipitate was resuspended in 4 mL of 0.15 M Tris-HCl (Ph=7.6) and centrifuged again at 100000 g for 60 min at 4 °C. The precipitate was then resuspended in 0.25 M sucrose at a 1:2 (w / v) ratio, resulting in 2 mL of microsome suspension per gram of liver tissue. The suspension was aliquoted and stored overnight in liquid nitrogen, then transferred to -80 °C for long-term storage the following day. All operations were performed in an ice bath. The microsomal protein content (mg / mL) was determined using the Bradford method.

[0039] 2. Determination of CYP2E1 activity in mouse liver microsomes Chlorzoxazone was selected as the probe substrate, and each reaction was performed in triplicate. The pre-incubation system for activity determination consisted of 0.5 mg / mL liver microsomes, 300 μM chlorzoxazone, and 100 mM phosphate buffer (pH 7.4). After mixing, the prepared microsome incubation system was incubated at 37°C for 5 min. Then, 1 mM NADPH was added to start the reaction, and after incubation for 30 min, 1 mL of ethyl acetate was added immediately to terminate the reaction. The mixture was then vortexed for 3 min, centrifuged at 12000 rpm for 10 min, and 800 μL of the upper organic phase was transferred to another clean centrifuge tube and dried under nitrogen at 37°C. 100 μL of the mobile phase was reconstituted, and 20 μL was injected as the sample.

[0040] 3. Sample processing Preparation of standard curve: Accurately weigh 18.5 mg of 6-OH CZX and add 1 mL of methanol to prepare a 100 mM 6-OH CZX stock solution. Dilute the stock solution with methanol to prepare working solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μM. Take the incubation system without NADPH as a blank control and add 10 μL of the above different concentrations of 6-OH CZX working solution to make standard curve samples with concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μM, respectively. Inject the samples according to the steps described in "2. Determination of CYP2E1 activity in mouse liver microsomes". The chromatographic column is a Kromasil 100-5-C18 column (4.6 × 250 mm), and the mobile phase is water:methanol = 44: 56. Isocratic elution was used at a flow rate of 1 mL / min, a column temperature of 35℃, a detection wavelength of 287 nm, and an injection volume of 20 μL. The standard curve equation was obtained by linear fitting with the final sample peak area (A) as the x-axis and the sample concentration (C) as the y-axis.

[0041] 4. Pharmacokinetic experiments in different mouse models Twelve 6-8 week old C57BL / 6 mice were randomly divided into two groups. One group received intraperitoneal injection of 75 mg / kg INH for 10 consecutive days, while the other group received intraperitoneal injection of physiological saline. Pharmacokinetic experiments were performed 24 hours after the last INH withdrawal. Five 6-8 week old C57BL / 6 mice were also divided into a control group and a Q11 group for subsequent pharmacokinetic experiments. Cyp2e1 knockout mice were obtained by Beijing Biocytogen Gene Biotechnology Co., Ltd. using conventional CRISPR / Cas9 technology. After being identified as homozygous (Cyp2e1- / -) knockout mice by qPCR, they were used for subsequent pharmacokinetic experiments. Five control mice and four Cyp2e1 knockout mice were divided into a control group and a Cyp2e1- / - group for subsequent pharmacokinetic experiments. Eleven C57BL / 6 mice were also divided into two groups. One group received intraperitoneal injection of CCl4 (diluted with olive oil to a 20% solution) at a dose of 5 mL / kg, twice a week for 6 weeks. The control group was given olive oil in the same manner. Pharmacokinetic studies were conducted after 6 weeks. The following procedures were followed for subsequent pharmacokinetic experiments: Before the experiment, an appropriate amount of CZX was weighed and a 6 mg / mL CZX solution was prepared. The drug was administered via tail vein injection at a volume of 10 mL / kg. Blood samples were collected from the orbital venous plexus at 2, 7, 15, 30, 45, and 60 minutes after tail vein administration. The samples were placed in heparin anticoagulant tubes and immediately centrifuged at 12000 rpm for 10 minutes. The supernatant was used for analysis or frozen at -80℃ for later testing.

[0042] 5. Rat pharmacokinetic experiments Ten healthy SD rats were divided into two groups of five each and fasted for 12 hours. Before the experiment, an appropriate amount of CZX was weighed and a 3 mg / mL CZX solution was prepared. The rats were administered the solution via gavage at a volume of 10 mL / kg. Blood samples were collected from the orbital venous plexus at 2, 7, 15, 30, 60, 90, 180, and 270 minutes after administration. The samples were placed in heparin anticoagulant tubes and centrifuged at 12,000 rpm for 10 minutes. The supernatant was used for analysis or frozen at -80℃ for later testing.

[0043] 6. Pharmacokinetic Experiments in Healthy Volunteers Twenty-nine healthy volunteers had not taken any medications within the month prior to the trial, had no prior medical history, and had normal liver and kidney function and electrocardiogram results. They fasted for 12 hours before the trial and took 600mg of chlorzoxazone tablets orally with 200ml of warm water the following morning. They were allowed to eat a standard meal 3 hours after taking the medication. Two mL of blood was collected venously at 0 hours before medication (0h) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours after medication. The blood samples were anticoagulated with heparin, centrifuged at 4500 rpm for 10 min, and the plasma was separated and stored at -80℃ for later analysis.

[0044] 7. Plasma Sample Processing Accurately pipette 20 μL of plasma sample into a 1.5 mL EP tube, add 20 μL of β-glucuronidase solution (final concentration: 5000 U / mL), incubate in a 37℃ water bath for 30 min, immediately add 1 mL of ethyl acetate, vortex mix for 3 min, centrifuge at 12000 rpm for 10 min, then pipette 800 μL of the upper organic phase into another clean centrifuge tube, dry under nitrogen at 37℃, reconstitute with 100 μL of mobile phase, and inject 20 μL of the mobile phase.

[0045] 8. Statistical Analysis SPSS 27.0 software was used for statistical analysis of the data. Pharmacokinetic parameters were calculated using DAS 2.0 software. For normally distributed data, t-tests were used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and Spearman's correlation test was used for correlation analysis. For non-normally distributed data, Mann-Whitney U test was used for comparisons between two groups, Kruskal-Wallis test was used for comparisons among multiple groups, and Spearman's correlation test was used for correlation analysis. A p-value < 0.05 was considered statistically significant.

[0046] This embodiment can employ an integrated CYP2E1 metabolic activity detection system, including: The sample processing module is used to perform step 3 of this embodiment: adding β-glucuronidase for incubation; extracting with ethyl acetate and drying with nitrogen; redissolving in the mobile phase and detecting the concentration by LC-MS / MS.

[0047] The ratio calculation module is used to execute step 6 of this embodiment to calculate and output the CYP2E1 metabolic activity based on the detected concentration according to the following formula; .

[0048] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A simple method for characterizing CYP2E1 activity for non-diagnostic purposes, characterized in that, The method comprises the following steps: a. obtaining a biological sample after administration of chlorzoxazone; b. determining the concentration ratio of 6-hydroxychlorzoxazone to chlorzoxazone at the highest point of the absorption phase of the sample; the highest point of the absorption phase is the time at which the concentration of 6-hydroxychlorzoxazone reaches a peak.

2. The method of claim 1, wherein, The biological sample is plasma or serum.

3. The method of claim 1, wherein, The highest point of the absorption phase is 5-20 minutes after administration in mice, 5-30 minutes in rats, and 0.5-2 hours in humans. Preferably, the highest point of the absorption phase is 10-20 minutes after administration in mice, 10-20 minutes in rats, and 1-2 hours in humans.

4. The method of claim 1, wherein, The dose of chlorzoxazone administered in step a. is in the range of 10-100 mg / kg in three species; Preferably, the dose of chlorzoxazone administered in step a. is 50-70 mg / kg in mice, 20-40 mg / kg in rats, and 5-15 mg / kg in humans. Further preferably, the dose of chlorzoxazone administered in step a. is 60 mg / kg in mice, 30 mg / kg in rats, and 10 mg / kg in humans.

5. The method of claim 1, wherein, The concentration ratio is calculated by the following formula: 。 6. The method of claim 1, wherein, Step a. is performed in a model suitable for investigating changes in CYP2E1 metabolic activity; the changes in activity include up-regulation of activity, down-regulation of activity, and no change in activity.

7. The method of claim 6, wherein, The model includes at least one of the following: a model of increased CYP2E1 activity induced by isoniazid, and a model of decreased CYP2E1 activity by CYP2E1 gene knockout, inhibitor treatment, and carbon tetrachloride treatment.

8. The method of claim 1, wherein, The biological sample is obtained by the following processing steps: incubation with β-glucuronidase; nitrogen blowing after ethyl acetate extraction; reconstitution with mobile phase and detection of concentration by LC-MS / MS.

9. A system for detecting CYP2E1 metabolic activity, characterized by It comprises: a sample processing module for performing the steps of claim 8; a ratio calculation module for performing the formula calculation of claim 5.

10. A kit for detecting CYP2E1 metabolic activity, characterized in that, It comprises a chlorzoxazone preparation, a sample processing kit, and a detection kit according to the method of any one of claims 1-8; the chlorzoxazone preparation is an injection solution with a concentration of 1-100 mg / mL; the sample processing kit comprises β-glucuronidase, ethyl acetate, and reconstitution mobile phase; the detection kit comprises standard samples of 6-hydroxychlorzoxazone and chlorzoxazone for LC-MS / MS.

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