Metabonomics detection method of irinotecan

Abstract

The invention discloses a metabonomics detection method of irinotecan, which is characterized in that irinotecan and metabolites thereof, namely 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G), are taken as research objects, camptothecin (CPT) is taken as an interior label, and a rapid and efficient UPLC-TQ-MS / MS method capable of synchronously detecting is established. According to UPLC-TQ-MS / MS results, as the dosage of irinotecan injected into the abdominal cavity of a mouse is increased, the content of irinotecan and the content of active metabolites of irinotecan in each tissue are gradually increased along with increase of the dosage of irinotecan injected into the abdominal cavity of the mouse. From a tissue distribution result, an affected organ of irinotecan and an active metabolite SN-38 thereof is mainly an intestinal tissue, and a colon is a main involved organ. The invention provides a scientific basis for the research of irinotecan metabolism, especially the research of adverse reaction of the irinotecan in the intestinal tract.

Description

Technical Field

[0001] The present invention relates to drug metabolism research, and in particular to a metabolomics detection method for irinotecan. Background Art

[0002] Irinotecan, one of the most commonly used chemotherapy drugs for colorectal cancer, can induce side effects such as intestinal mucositis. Particularly in the context of colorectal cancer, severe gastrointestinal reactions limit the dosage of irinotecan, preventing it from reaching a therapeutic dose and achieving satisfactory therapeutic results.

[0003] The literature generally believes that the mechanism of toxicity is related to the accumulation of SN-38 in the intestine.

[0004] Irinotecan is a widely used chemotherapy drug for the treatment of colorectal cancer. Its efficacy and toxicity are closely related to its metabolism in vivo. Irinotecan, a camptothecin analogue with higher water solubility than camptothecin, belongs to the class of topoisomerase inhibitors. It exerts its anticancer effects by inhibiting cancer cell proliferation by affecting the DNA structure and function of cancer cells. Irinotecan is a prodrug that is metabolically activated to SN-38 in vivo. A growing number of studies have revealed the relationship between irinotecan metabolism and its toxic side effects. Irinotecan is converted to the active metabolite SN-38 by carboxylesterase 2 (CES2), which is further metabolized to the inactive metabolite SN-38G. Irinotecan exhibits broad antitumor activity both in vitro and in vivo and has a more predictable and clinically manageable toxicity profile than the initially isolated structure.

[0005] Chromatographic analysis, with its exceptional sensitivity, selectivity, accuracy, and precision, meets the needs of most drug testing. With the continuous advancement of chromatographic techniques and the widespread adoption of instrumentation, high-performance liquid chromatography (HPLC) and its coupled technologies (such as LC-MS and LC-MS / MS) have become the primary method for analyzing and detecting drugs and their metabolites in in vivo samples. UPLC-TQ-MS / MS is an efficient, sensitive, and versatile quantitative analysis technique that utilizes multiple reaction monitoring (MRM) mode to effectively analyze complex chemical components. This method significantly improves analytical efficiency and accuracy and is a key tool for the quantitative detection and analysis of complex components in biological samples. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to develop and validate a stable and sensitive UPLC-TQ-MS / MS method for quantifying the concentrations of irinotecan and its metabolites (7-ethyl-10-hydroxycamptothecin SN-38 and SN-38 glucuronide SN-38G). Irinotecan and its metabolites SN-38 and SN-38G were studied, with camptothecin (CPT) serving as the internal standard. By optimizing chromatographic separation conditions and mass spectrometry parameters, highly sensitive and specific detection of irinotecan and its major metabolites was achieved, providing important data support for a deeper understanding of its tissue-specific toxicity.

[0007] Technical solution: In order to achieve the above objectives, the technical solution adopted by the present invention is: A metabolomics detection method for irinotecan comprises the following steps: (1) Preparation of mixed reference solution and internal standard solution Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare reference stock solutions of certain concentrations. Take each reference substance stock solution and dilute it with methanol to prepare a series of concentrations of mixed reference substance solutions; Camptothecin was added with methanol to prepare an internal standard solution; (2) Preparation of standard curve Mixed reference solutions containing internal standard solutions at different concentrations were injected into UPLC-TQ-MS / MS for analysis. The standard curve equation was drawn with the concentration of the mixed reference solution as the abscissa and the ratio of the peak area of the reference solution to the peak area of the internal standard solution as the ordinate. (3) Experimental animal modeling The experimental mice were fed adaptively for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg kg -1 Azomethane (AOM), one week later, the model group mice drank 2.0%-2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, for three consecutive cycles; (4) Sample processing: After modeling, except for the control group, the remaining C57BL / 6 mice that had undergone AOM / DSS modeling were randomly divided into a model group and different irinotecan concentration groups according to body weight. The drug administration cycle was six days. During the drug administration period, the mice in the control and model groups were intraperitoneally injected with an equal volume of normal saline. Blood was collected from the mouse orbits and then sacrificed by cervical dislocation. The liver, kidney, spleen, testicle, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Serum samples: Serum samples were placed in blank centrifuge tubes. Proteins were precipitated with a formic acid-methanol solution containing a certain amount of camptothecin, vortexed, and all samples were allowed to stand for a while before centrifugation. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation to dryness. Tissue samples: Accurately weigh liver, kidney, spleen, testis, jejunum, ileum, and colon tissues. Add a certain amount of formic acid-methanol solution containing camptothecin to the above samples to precipitate protein. Vortex and let all samples stand for a while. Centrifuge and transfer the supernatant to a clean centrifuge tube. Concentrate and evaporate to dryness in a vacuum centrifuge. (5) Metabolite analysis Serum and tissue samples were reconstituted with cold methanol, vortexed, and centrifuged. The supernatant was placed in an intubation tube for UPLC-TQ-MS / MS analysis.

[0008] As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the preparation method of the mixed reference solution and internal standard solution in step (1) is: Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare a concentration of 1 mg. mL -1 Reference substance stock solution; A series of mixed reference solutions were prepared by diluting each reference stock solution with methanol. The irinotecan concentrations were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 ; The concentrations of 7-ethyl-10-hydroxycamptothecin were: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 ; The concentrations of SN-38 glucuronide were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 ; Dissolve camptothecin in methanol and add it to each mixed reference solution as an internal standard. The concentration of camptothecin in each mixed reference solution is 250 ng / mL. As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the experimental animal modeling method in step (3) is: The mice were fed adaptively for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg kg -1 Azomethane (AOM), one week later, the mice in the model group drank 2.0%~2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, for 3 consecutive cycles.

[0009] As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the sample processing method in step (4) is: After the modeling was completed, the mice were sacrificed by orbital blood sampling and then dissected by cervical dislocation. The liver, kidney, spleen, testis, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Serum sample: 50 μL serum sample was placed in a blank centrifuge tube, and the above sample was mixed with 350 μL 0.1% formic acid methanol solution (containing 250 ng mL -1 The protein was precipitated with camptothecin and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness. Tissue samples: 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues were accurately weighed and 350 μL of 0.1% formic acid methanol solution (containing 250 ng mL -1 The protein was precipitated with camptothecin and vortexed. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min for 10-15 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness. As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the chromatographic conditions for the preparation of the standard curve in step (2) and the UPLC-TQ-MS / MS analysis of the metabolites in step (5) are as follows: Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase consisted of 0.1% formic acid in water (phase B) and acetonitrile (phase A) for gradient elution. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35°C, the autosampler temperature was 5°C, and the analysis time was 5.5 min. The gradient elution program was as follows: Time / min A% B% 0-0.5 10 90 0.5-2.5 10-45 90-55 2.5-4.0 45 55 4.0-5.0 45-90 55-10 5.0-5.5 90-10 10-90 The mass spectrometry conditions were as follows: electrospray ionization positive ion source (ESI +) and multiple reaction monitoring (MRM) scan mode for quantification, with a capillary voltage of 3.0 kV, a desolvation temperature of 350 °C, and a conical gas flow rate of 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas was argon.

[0010] Beneficial effects: The metabolomics detection method for irinotecan provided by the present invention has the following advantages compared with the prior art: To effectively separate irinotecan, SN-38, SN-38G, and CPT while minimizing matrix effects, this study employed an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a gradient elution using a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Based on the response and extraction recovery of each component, 0.1% formic acid in methanol and 50% methanol were selected as the precipitant and resolvation solvent, respectively, during sample processing. Irinotecan and its active metabolite, SN-38, exist as a lactone (active) and a carboxylate (inactive) form, and the equilibrium between these two forms is pH-dependent, with acidic pH favoring the formation of the active lactone form. At pH 9, the lactone form rapidly converts to the carboxylate form, necessitating the use of acidified methanol as the protein precipitant. The chromatogram demonstrates good separation of the components within 5.5 minutes, a run time that allows for the analysis of a large number of samples within a reasonable timeframe. For mass spectrometry conditions, positive ion mode was selected because it provides a higher intensity of precursor ion peaks comparable to existing literature methods. No impurities or endogenous substances that significantly interfered with the determination of the components and internal standard were found in the blank matrix, and no significant matrix effects were found. A systematic methodological investigation showed that the UPLC-TQ-MS / MS analysis method established in this experiment had good linear relationships among the components ( r 2 >0.99), precision, accuracy, extraction recovery, matrix effect, and stability can all meet the determination requirements of biological samples and lay the foundation for subsequent experiments.

[0011] This study investigated irinotecan and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G), using camptothecin (CPT) as an internal standard. A rapid, efficient, and simultaneous UPLC-TQ-MS / MS method was developed. UPLC-TQ-MS / MS results revealed that the levels of irinotecan and its active metabolites in various tissues increased with increasing doses of intraperitoneal irinotecan in mice. Tissue distribution revealed that irinotecan and its active metabolite, SN-38, were primarily affected in the intestine, with the colon being the primary target.

[0012] The invention provides a scientific basis for the study of irinotecan metabolism, especially for the study of its adverse reactions in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows the MRM chromatograms of CPT-11, SN-38, SN-38G, and CPT.

[0014] Figure 2 shows the UPLC-TQ-MS / MS chromatograms of irinotecan and its metabolites in serum. Blank serum (A), blank serum with standard solution (B), and intraperitoneal injection of 40 mg / kg irinotecan (C).

[0015] Figure 3. Contents of irinotecan and its metabolites in serum and testis ( ± SEM , n = 6).

[0016] Figure 4 Contents of irinotecan and its metabolites in kidney and spleen ( ± SEM , n = 6).

[0017] Figure 5 Contents of irinotecan and its metabolites in the liver and jejunum ( ± SEM , n = 6).

[0018] Figure 6 Contents of irinotecan and its metabolites in the ileum and colon ( ± SEM , n = 6). DETAILED DESCRIPTION

[0019] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0020] Example 1 1 Experimental Materials 1.1 Experimental Animals experimental animals Forty-two 8-week-old SPF C57BL / 6 male mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (certificate number SCXK (Beijing) 2021-0006). The animal experiments were approved by the Ethics Committee of Shaanxi University of Chinese Medicine, and animal husbandry and experimental procedures complied with the regulations for experimental management.

[0021] 1.2 Drugs and reagents Irinotecan (CPT-11, ≥98%, batch number: O21HB198258), 7-ethyl-10-hydroxycamptothecin (SN-38, ≥98%, batch number: H10D9Y76874), SN-38 glucuronide (SN-38G, ≥98%, batch number: JB248688), and camptothecin (CPT, ≥98%, batch number: L26M7X11954) were purchased from Shanghai Yuanye Company. Formic acid (LC-MS, product number: 5.33002), methanol (LC-MS, product number: 1.06035), and acetonitrile (LC-MS, product number: 1.00029) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0022] 1.3 Instruments and Equipment Table 1 Instrument and equipment information Instruments and Equipment Manufacturer Microfuge 20R Benchtop High-Speed Refrigerated Centrifuge Beckman Coulter, Inc. Acquity UPLC I-class + Xevo TQ-XS Waters Technology (Shanghai) Co., Ltd. Fully automatic sample rapid grinding instrument Shanghai Jingxin Industrial Development Co., Ltd. Vortex mixer Tiangen Biochemical Technology Co., Ltd. Mettler MS105DU Electronic Analytical Balance Mettler-Toledo AG of Switzerland Mettler ME204 electronic balance Mettler-Toledo AG of Switzerland Labconco CentriVap Refrigerated Benchtop Vacuum Centrifuge Concentrator Labconco, USA 2 Experimental methods 2.1 Sample processing The mice were fed adaptively for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg kg -1 Azomethane (AOM), one week later, the mice in the model group drank 2.0%~2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, for 3 consecutive cycles.

[0023] After the modeling was completed, except for the control group, the remaining C57BL / 6 mice that had undergone AOM / DSS modeling were randomly divided into 6 groups according to their body weight, namely the model group (Model) and five irinotecan dosage groups (CPT-11 5 mg kg -1 、CPT-11 10 mg kg -1 、CPT-11 20 mg kg -1 、CPT-11 40 mg kg -1 、CPT-11 80 mg kg -1 The dosing cycle was six days. The weight of the mice was recorded before each day of the experiment, and the mice were observed for diarrhea. The control group and the model group mice were intraperitoneally injected with equal volumes of normal saline during the dosing period. The five irinotecan dose groups were each intraperitoneally injected with a dose of 5 mg kg -1 , 10 mg kg -1 , 20 mg kg-1 , 40 mg kg -1 , 80 mg kg -1 After blood was collected from the mice's orbits, they were killed by cervical dislocation and dissected. The liver, kidney, spleen, testicles, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Serum sample: 50 μL serum sample was placed in a blank centrifuge tube, and the above sample was mixed with 350 μL 0.1% formic acid methanol solution (containing 250 ng mL -1 Protein was precipitated by centrifugation with CPT (CPT) and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness.

[0024] Tissue samples: 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues were accurately weighed and 350 μL of 0.1% formic acid methanol solution (containing 250 ng mL -1 Protein was precipitated by centrifugation with CPT and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness.

[0025] Before analysis, all samples were reconstituted with 100 μL of 50% cold methanol, vortexed for 3 minutes, and centrifuged at 13,000 rpm / min at 4°C for 10 minutes. The supernatant was then transferred to an insert for UPLC-TQ-MS / MS analysis. Data were analyzed using MassLynx software.

[0026] 2.2 Preparation of reference solution and internal standard solution CPT-11, SN-38, SN-38G, and CPT standards were accurately weighed and dissolved in methanol to prepare a stock solution at a concentration of 1 mg / mL. The solution was then stored in a refrigerator at 4°C until use. A series of mixed standard solutions were prepared by diluting with methanol, including 10 concentration points: CPT-11 concentrations of 10,000, 5,000, 2,500, 1,250, 625, 312.5, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 SN-38 concentrations were: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 The concentrations of SN-38G were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 Camptothecin was dissolved in methanol and added to each mixed reference solution as an internal standard. The concentration of camptothecin was 250 ng / mL. The concentrations of quality control samples (QCs) were: 2.44, 4.88, 156, and 8000 ng for CPT-11. mL -1 , SN-38 is 2.44, 4.88, 78, 4000 ng mL -1 SN-38G: 2.44, 4.88, 156, 8000 ng mL -1 All standard solutions were stored in a refrigerator at 4°C before analysis.

[0027] 2.3 Chromatographic conditions Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase consisted of 0.1% formic acid in water [v / v] (B)-acetonitrile (A) for gradient elution. The gradient elution program is shown in Table 2. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35°C, the autosampler temperature was 5°C, and the analysis time was 5.5 min.

[0028] Table 2 Mobile phase gradient elution program Time (min) Flow rate (mL / min) A% B% 0-0.5 0.3 10 90 0.5-2.5 0.3 10-45 90-55 2.5-4.0 0.3 45 55 4.0-5.0 0.3 45-90 55-10 5.0-5.5 0.3 90-10 10-90 2.4 Mass spectrometry conditions Electrospray ionization (ESI) + ) and multiple reaction monitoring (MRM) scan mode for quantification. The capillary voltage was 3.0 kV, the desolvation temperature was 350 °C, and the conical gas flow rate was 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas is argon.

[0029] 2.5 Methodological Review According to the requirements of the FDA and the technical guidelines for clinical pharmacokinetic studies of chemical drugs, the methodological investigation included the specificity and linearity, precision and accuracy, matrix effect and extraction recovery, and stability of CPT-11 and its metabolites.

[0030] 2.5.1 Specificity Investigation and Linear Relationship Compare the MRM chromatograms of blank serum and blank serum plus mixed standards to examine the specificity of the method. Blank serum is mainly used to determine whether endogenous substances interfere with the analytical method, while the internal standard is mainly used to evaluate whether the internal standard interferes with the determination of the sample or whether its interference is within the acceptable range of the analytical method. Take 50 μL of blank serum in a centrifuge tube, add 350 μL of methanol solution to precipitate protein, treat according to the sample treatment method of "2.1", and operate and inject according to the chromatography and mass spectrometry conditions of "2.3" and "2.4" to obtain the blank serum chromatogram. Take 10 μL of the mixed standard curve solution C1-C10 respectively, treat according to the sample treatment method of "2.1", and operate and inject according to the corresponding chromatography and mass spectrometry conditions. The concentration of the mixed standard solution is the horizontal axis ( X , ng / mL), the ratio of the standard peak area to the internal standard peak area is the vertical axis ( Y ) to draw a standard curve, and the weight coefficient of the linear regression equation 1 / Y The lowest concentration point of the standard curve was taken as the minimum quantification limit ( LLOQ ), and the minimum quantification limit should meet the precision ≤ 20%, accuracy ( RE% ) between -20% and 20%.

[0031] 2.5.2 Precision and Accuracy Assessment Take 50 μL of blank serum and 10 μL of quality control samples of high, medium and low concentrations (the concentrations are QC1-LOQ; QC2-within 3 times of LOQ; QC3-√QC2*QC4; QC4-80%UOQ) and process them according to the sample processing method under "2.1". Repeat 6 times for each concentration and measure 3 batches continuously. The concentration of quality control samples is obtained by substituting it into the linear regression equation of the standard curve and compared with the concentration of the prepared reference substance. The intra-day and inter-day precision and accuracy of the QC samples are calculated, and the relative standard deviation is used respectively. RSD% and relative standard error RR% Come and evaluate.

[0032] 2.5.3 Extraction recovery and matrix effect A certain amount of quality control sample was added to the blank serum sample and processed according to the sample processing method in "2.1" above (A). 50 μL of blank serum was first added with methanol to precipitate protein, and then CPT-11, SN-38, and SN-38G at the same concentration as after treatment with sample A were added (B). Another aliquot was dissolved in pure methanol solution and diluted to the same concentration as after treatment with sample A (C). Matrix effect and extraction recovery were calculated using the peak area ratios of A / B and A / C, respectively.

[0033] 2.5.4 Stability investigation Stability assessments of stock and working solutions of analytes, internal standards, and QC samples should be conducted under various storage conditions, and the timescale should take into account the actual storage duration of the samples. Sample stability includes both short-term and long-term stability. Short-term stability typically assesses the stability of analyte and internal standard stock and working solutions, as well as QC samples, from refrigerated storage to room temperature or sample processing temperature, freeze-thaw stability of QC samples, and stability of processed samples at autosampler temperature. These are all critical to the accuracy and reproducibility of test results. Short-term stability assessments are conducted by storing QC samples at room temperature for 8 hours, at -80°C overnight, and in a sample tray for 24 hours. Long-term stability assessments involve the storage stability of QC samples throughout the analytical process, as well as the long-term storage stability of analyte and internal standard stock and working solutions. These factors also impact the accuracy and reliability of analytical results. The long-term stability of QC samples is assessed by storing them at -80°C for one week.

[0034] 2.5.5 Statistical analysis The experimental data are all based on ± SEM Statistical analysis was performed using GraphPad Prism 8.3.0. t test, P <0.05 was statistically significant.

[0035] 3 Experimental Results 3.1 Optimization of mass spectrometry conditions Using the MRM mode, the cone voltage and collision energy of the test compound and the internal standard compound were optimized. The mass spectrometry optimization conditions are shown in Table 3. Representative chromatograms are shown in Table 3. Figure 1 shown.

[0036] Table 3 Mass spectrometry optimization conditions Analyte tR (min) Precursor Ion (m / z) Daughter Ion (m / z) Cone voltage (V) Collision energy (eV) Ionization mode CPT-11 2.90 587.52 124.03 50.0 32.0 <![CDATA[ES + ]]> SN-38 3.36 393.30 249.11 10.0 46.0 <![CDATA[ES + ]]> SN-38G 2.65 569.55 393.23 10.0 26.0 <![CDATA[ES + ]]> CPT 3.54 349.19 219.02 8.0 48.0 <![CDATA[ES + ]]> 3.2 Methodological Review 3.2.1 Specificity Investigation and Linear Relationship Under the experimental conditions, irinotecan and its metabolites SN-38, SN-38G, and internal standard CPT had good separation and strong peak signals. No significant interference peaks were observed at the retention times of the analytes and internal standards in the blank matrix, indicating that the UPLC-TQ-MS / MS method established in this experiment is specific. The retention times of CPT-11, SN-38, SN-38G, and CPT were 2.88, 3.35, 2.64, and 3.53 min, respectively. kg-1 The chromatogram after irinotecan is as follows Figure 2 shown.

[0037] The regression equation, linear coefficient, and linear range of irinotecan and its metabolites SN-38 and SN-38G in serum are shown in Table 4. The regression equation of irinotecan and its metabolites SN-38 and SN-38G has a good linear coefficient ( r 2 >0.99), the lowest concentration of the analyte that can be detected by this analytical method is defined as the lower limit of quantification, so the lower limit of quantification for CPT-11, SN-38, and SN-38G is 2.44 ng. mL -1 , which meets the requirements of tissue distribution experiments.

[0038] Table 4 Linear range, regression equation and linear coefficient of irinotecan and its metabolites

[0039] 3.2.2 Precision and accuracy The intraday ( n =6) and daytime ( n =6) were investigated for accuracy and precision, and the results are shown in Table 5. The degree of closeness between the measured value and the true value is within ± 20%, and the degree of deviation between the measured value and the average value ( RSD ,%) was lower than 15%, indicating that the precision and accuracy of this method met the requirements for biological sample determination.

[0040] Table 5 Inter-day and intra-day accuracy and precision of analytes in serum

[0041] 3.2.3 Extraction recovery and matrix effect The extraction recoveries and matrix effects of CPT-11 and its metabolites, SN-38 and SN-38G, in serum are shown in Table 6. Matrix effects for CPT-11, SN-38, and SN-38G ranged from 94.81% to 97.94%, 91.96% to 97.57%, and 94.81% to 97.93%, respectively. This indicates that this assay effectively avoids matrix effects caused by endogenous substances in the matrix. The extraction recoveries for CPT-11, SN-38, and SN-38G were 88.79% to 97.88%, 90.36% to 98.40%, and 88.79% to 97.89%, respectively. The sample preparation process was satisfactory and enabled accurate determination of the analytes in biological samples.

[0042] Table 6 Matrix effects and extraction recoveries of analytes in serum .

[0043] 3.2.4 Stability investigation The stability of the analytes, including short-term stability and long-term stability, was investigated using quality control samples. The results are shown in Tables 7 and 8. The measured values were close to the true values within ± 15%, and the deviations from the mean values ( RSD , %) is lower than 15%, which meets the requirements for biological sample determination.

[0044] Table 7 Stability of analytes in serum (recovery, %) n =6)

[0045] Table 8 Stability of analytes in serum (recovery, %) n =6)

[0046] 3.3 Tissue distribution of irinotecan in mice The concentrations of irinotecan and its metabolites SN-38 and SN-38G in mouse serum, testis, kidney, spleen, liver, jejunum, ileum, and colon were determined. Figures 3 to 6 Most of the samples measured were within the linear range of the analyte. Irinotecan is abundant in small intestinal samples, especially in colonic tissue, where concentrations in some samples were outside the linear range. It was necessary to dilute the samples with higher concentrations to correctly quantify the samples.

[0047] This study used irinotecan and its metabolites SN-38 and SN-38G as the research subjects, using CPT as the internal standard, to develop a rapid, efficient, and synchronously detectable UPLC-TQ-MS / MS method. UPLC-TQ-MS / MS results revealed that the levels of irinotecan and its active metabolites in various tissues increased with increasing doses of intraperitoneal irinotecan in mice. Tissue distribution revealed that irinotecan and its active metabolite SN-38 were primarily affected in the intestine, with the colon being the primary affected organ. This study provides a scientific basis for the study of irinotecan and its metabolism.

Claims

1. A metabolomics detection method for irinotecan, characterized in that: The following steps are included: (1) Preparation of mixed reference solution and internal standard solution Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare reference stock solutions of certain concentrations. Take each reference substance stock solution and dilute it with methanol to prepare a series of concentrations of mixed reference substance solutions; Take camptothecin and add methanol to prepare the internal standard solution; (2) Preparation of standard curve Mixed reference solutions containing internal standard solutions at different concentrations were injected into UPLC-TQ-MS / MS for analysis, with the concentration of the mixed reference solution as the abscissa and the ratio of the reference peak area to the internal standard peak area as the ordinate to draw the standard curve equation; (3) Experimental animal modeling The experimental mice were adaptively fed for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of azomethane (AOM). One week later, the mice in the model group drank dextran sulfate sodium (DSS) solution for 7 days and normal drinking water for 14 days, and this cycle lasted for 3 consecutive days. (4) Sample processing: After modeling, except for the control group, the remaining C57BL / 6 mice induced by AOM / DSS modeling were randomly divided into model groups according to body weight (different irinotecan concentrations; the administration cycle was six days. During the administration period, the mice in the control and model groups were intraperitoneally injected with equal volumes of normal saline. Blood was collected from the mice's orbits and then sacrificed by cervical dislocation for dissection. The liver, kidney, spleen, testicle, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Serum samples: Serum samples were placed in blank centrifuge tubes. Proteins were precipitated with a formic acid-methanol solution containing a certain amount of camptothecin, vortexed, and all samples were allowed to stand for a while before centrifugation. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation to dryness. Tissue samples: Accurately weigh liver, kidney, spleen, testis, jejunum, ileum, and colon tissues. Add a certain amount of formic acid-methanol solution containing camptothecin to the above samples to precipitate protein. Vortex and let all samples stand for a while. Centrifuge and transfer the supernatant to a clean centrifuge tube. Concentrate and evaporate to dryness in a vacuum centrifuge. (5) Metabolite analysis Serum and tissue samples were reconstituted with cold methanol, vortexed, and centrifuged. The supernatant was placed in an intubation tube for UPLC-TQ-MS / MS analysis.

2. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The preparation method of the mixed reference solution and internal standard solution in step (1) is as follows: Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare a concentration of 1 mg. mL -1 Reference substance stock solution; Take each reference substance stock solution and dilute it with methanol to prepare a series of mixed reference substance solutions; Irinotecan concentrations were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 4.88, 2.44 ng mL -1 ; The concentrations of 7-ethyl-10-hydroxycamptothecin were: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 ; The concentrations of SN-38 glucuronide were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 4.88, and 2.44 ng. mL -1 ; Camptothecin was dissolved in methanol and added to each mixed reference solution as an internal standard. The concentration of camptothecin was 250 ng / mL.

3. The metabolomics detection method of irinotecan according to claim 1, characterized in that: The experimental animal modeling method in step (3) is: The mice were fed adaptively for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg kg -1 Azomethane (AOM), one week later, the mice in the model group drank 2.0%~2.5% DSS (dextrose sodium sulfate) solution for 7 days and normal drinking water for 14 days, for 3 consecutive cycles.

4. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The sample processing method in step (4) is: After the modeling was completed, the mice were sacrificed by orbital blood sampling and then dissected by cervical dislocation. The liver, kidney, spleen, testis, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Serum sample: 50 μL serum sample was placed in a blank centrifuge tube, and the above sample was mixed with 350 μL 0.1% formic acid methanol solution (containing 250 ng mL -1 The protein was precipitated with camptothecin and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness. Tissue samples: 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues were accurately weighed and 350 μL of 0.1% formic acid methanol solution (containing 250 ng mL -1 All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min for 10 to 15 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness.

5. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The chromatographic conditions for the preparation of the standard curve in step (2) and the UPLC-TQ-MS / MS analysis in step (5) of the metabolite analysis were as follows: Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase consisted of 0.1% formic acid in water (phase B) and acetonitrile (phase A) for gradient elution. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35°C, the autosampler temperature was 5°C, and the analysis time was 5.5 min. The gradient elution program was as follows: 。 6. Mass spectrometry conditions: using electrospray ionization positive ion source (ESI + ) and multiple reaction monitoring (MRM) scan mode for quantification, with a capillary voltage of 3.0 kV, a desolvation temperature of 350 °C, and a conical gas flow rate of 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas was argon.

7. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The mass spectrometry conditions for each compound in step (5) are: 。 8. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The regression equation of step (2.3) is: 。

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