Method for detecting metabolite containing phosphate radical and application thereof

Abstract

The invention discloses a method for detecting metabolite containing phosphate radicals, which is characterized in that a mass spectrum label with stable and characteristic chlorine isotope distribution is introduced through a chlorination reagent derivatization strategy, so that a derivatization product can be subjected to rapid and high-specificity preliminary identification based on primary mass spectrum data. By combining with an automatic data mining program specially developed for this purpose, high-throughput and accurate screening and locking of potential phosphate radical-containing metabolites from massive and complex non-targeted metabonomics original data are realized, and the data analysis efficiency and reliability are greatly improved. The method is successfully applied to analysis of actual biological samples such as cholestasis disease models, efficient identification and accurate quantification of various key metabolites such as nucleotide and sugar phosphoric acid are achieved, disturbance of related metabolic pathways is disclosed, and a powerful innovative analysis tool is provided for disease mechanism research and biomarker discovery.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to a method for detecting phosphate-containing metabolites and its applications. Background Technology

[0002] Phosphate-containing metabolites are a vital class of endogenous compounds in living organisms, widely participating in and regulating many core life processes, including energy metabolism (such as ATP and ADP), cell signal transduction (such as IP3), genetic information transmission (such as nucleotides), and the regulation of the activity of various key enzymes. In metabolomics, exocomics, and disease mechanism research, achieving comprehensive and precise analysis of phosphate-containing metabolites in biological samples (such as blood, tissues, and cells) has significant scientific value and application prospects for revealing their physiological functions, discovering disease-related metabolic disturbances, and identifying potential biomarkers.

[0003] Currently, the analysis of phosphate-containing compounds mainly relies on the coupling of various chromatographic and detection techniques. Liquid chromatography-mass spectrometry (LC-MS) has become the mainstream platform for metabolite analysis in complex systems due to its high separation capability, high sensitivity, and strong structural resolution potential. However, the high polarity and strong hydrophilicity of phosphate groups result in very weak retention on commonly used reversed-phase chromatography, often eluting near the dead time, leading to poor separation and susceptibility to severe interference from co-eluting components in complex matrices. To improve retention, hydrophilic interaction chromatography has become a commonly used alternative, but it still suffers from problems such as long column equilibration time, poor peak shape, limited resolution, and potentially poor batch-to-batch reproducibility, posing challenges when analyzing structurally similar isomers (such as glucose-1-phosphate and glucose-6-phosphate).

[0004] Therefore, there is an urgent need in this field to develop a new analytical strategy that can effectively improve the chromatographic separation performance of phosphate-containing metabolites, and ultimately achieve efficient and rapid non-targeted discovery and identification of these key metabolites from complex systems. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for detecting phosphate-containing metabolites and its application.

[0006] According to one aspect of the present invention, a method for detecting phosphate-containing metabolites is provided, comprising the following steps: derivatizing the phosphate-containing metabolites in the sample to be tested with a chlorination reagent; performing liquid chromatography-mass spectrometry analysis on the derivatized sample to obtain mass spectrometry data; and identifying the derivatized phosphate-containing metabolites in the sample to be tested based on the characteristic isotope signal of chlorine in the chlorination reagent in the mass spectrometry data.

[0007] Preferably, the identification based on characteristic isotope signals specifically includes: screening precursor ions from the mass spectrometry data that simultaneously contain a mass-to-charge ratio (M) peak and an isotope (M+2) peak; calculating the peak intensity ratio of the M peak to the (M+2) peak; and identifying precursor ions whose peak intensity ratio is within a preset range as potential derivatized metabolites.

[0008] Preferably, the preset range is 2.75 to 3.25.

[0009] Preferably, the filtering and calculation steps are implemented using a Python program.

[0010] Preferably, the chlorination reagent derivatization treatment includes: mixing the pretreated sample with a first reaction solution containing carbodiimide and imidazole to carry out a first reaction; subsequently adding a second reaction solution containing a chlorination reagent and methylimidazole to carry out a second reaction; wherein the chlorination reagent is [5-(2-chlorophenyl)isoxazol-3-yl]methylamine.

[0011] Preferably, the conditions for the first reaction are: reacting at 40°C for 30 minutes; and the conditions for the second reaction are: reacting at 40°C for 2 hours.

[0012] Preferably, before the chlorination of the phosphate-containing metabolites, a sample pretreatment and enrichment step is included, wherein the enrichment step is performed using a weak anion exchange solid-phase extraction column.

[0013] Preferably, the sample pretreatment and enrichment steps specifically include: mixing biological tissue samples with physiological saline and pre-cooled methanol, homogenizing, centrifuging, collecting the supernatant and combining and concentrating it to obtain a residue; redissolving the residue in ammonium acetate buffer, and extracting it through an activated weak anion exchange solid-phase extraction column; rinsing with ammonium acetate buffer and methanol, and then eluting the target compound with ammonia-methanol solution to obtain the enriched sample.

[0014] Preferably, in the liquid chromatography-mass spectrometry analysis: the column packing material is phenyl-bonded silica gel; mobile phase A is a 5 mM ammonium bicarbonate aqueous solution, and mobile phase B is a 5 mM ammonium bicarbonate methanol-water solution, wherein the methanol volume concentration is 90%; a gradient elution program is used.

[0015] Preferably, the gradient elution program is as follows: 0-3.0 min, mobile phase A decreases from 92% to 80%, and mobile phase B increases from 8% to 20%; 3.0-6.5 min, mobile phase A decreases from 80% to 67%, and mobile phase B increases from 20% to 33%; 6.5-7.0 min, mobile phase A decreases from 67% to 45%, and mobile phase B increases from 33% to 55%; 7.0-9.0 min, mobile phase A decreases from 45% to 5%, and mobile phase B increases from 55% to 95%; 9.0-10.9 min, mobile phase A remains at 5%, and mobile phase B remains at 95%; 10.9-11.0 min, mobile phase A increases from 5% to 92%, and mobile phase B decreases from 95% to 8%. The mass spectrometry analysis is performed in positive ion mode using an electrospray ionization (ESI) source, and the scanning mode is full scan.

[0016] According to another aspect of the present invention, the above method is provided for use in rapid screening of phosphate-containing metabolites in non-targeted metabolomics analysis or in screening or studying biomarkers associated with cholestatic diseases.

[0017] This invention utilizes a derivatization strategy with the chlorinated reagent [5-(2-chlorophenyl)isoxazol-3-yl]methylamine (ClXIMA) to significantly enhance the retention and separation capabilities of highly polar phosphate-containing metabolites in reversed-phase chromatography. This effectively solves the common technical challenges of weak retention, poor peak shape, and difficulty in distinguishing isomers in conventional analysis of these compounds. Simultaneously, this derivatization reaction introduces a stable and characteristic chlorine isotope distribution into the metabolites. The mass spectrometry tagging of [database name] enables rapid and highly specific preliminary identification of derivatized products based on primary mass spectrometry data. Combined with a specially developed automated data mining program, this method achieves high-throughput and precise screening and identification of potential phosphate-containing metabolites from massive and complex untargeted metabolomics raw data, significantly improving data analysis efficiency and reliability. This method has been successfully applied to the analysis of real biological samples, such as cholestasis disease models, achieving not only efficient identification and accurate quantification of various key metabolites such as nucleotides and sugar phosphates, but also revealing perturbations in related metabolic pathways, providing a powerful and innovative analytical tool for disease mechanism research and biomarker discovery. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0019] Figure 1 This is a comparison chart of different chromatographic columns provided according to embodiments of the present invention.

[0020] Figure 2 This is a comparison chart of different mobile phase additives provided according to embodiments of the present invention.

[0021] Figure 3 This is a comparison diagram of the mixed standard before and after derivatization according to an embodiment of the present invention.

[0022] Figure 4 This is a comparison diagram of isomer derivatization before and after, according to an embodiment of the present invention.

[0023] Figure 5 This is a primary mass spectrum of the UMP-ClXIMA derivative provided in an embodiment of the present invention.

[0024] Figure 6 This is a comparison of first-stage mass spectra before and after ADP-Glc derivatization according to an embodiment of the present invention. Detailed Implementation

[0025] The following examples are provided to enable those skilled in the art to better understand the present invention. It should be noted that, unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] Example 1: Detection of phosphate-containing metabolites in a mixture of standards using ClXIMA derivatization combined with LC-MS

[0029] 1. Reagents and Instruments

[0030] To ensure the accuracy, reproducibility, and high sensitivity of the experiment, key specialized reagents, high-purity standards, and a high-resolution liquid chromatography-mass spectrometry (LC-MS) system required for the derivatization reaction were selected and identified. Reagents: [5-(2-chlorophenyl)isoxazol-3-yl]methylamine (ClXIMA, purity >95%, custom-synthesized); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), imidazole, 1-methylimidazole (all purchased from Sigma-Aldrich, purity >98%); chromatographic grade methanol, acetonitrile, and dimethyl sulfoxide (DMSO); analytical grade ammonium acetate and ammonia; and Milli-Q ultrapure water.

[0031] Reference standards: Take appropriate amounts of galactose-6-phosphate, mannose-6-phosphate, glucose-6-phosphate, 3-phosphoglyceric acid, ribose-5-phosphate, uridine diphosphate glucose, sucrose-6-phosphate, adenosine diphosphate glucose, glucose-1-phosphate, fructose-1,6-bisphosphate, guanosine monophosphate, inosine monophosphate, cytidine monophosphate, deoxyguanosine monophosphate, uridine monophosphate, deoxycytidine monophosphate, glucosamine-6-phosphate, adenosine monophosphate, thymidine monophosphate, N6-methyladenosine monophosphate, deoxyadenosine monophosphate, deoxyguanosine diphosphate, cytidine diphosphate, uridine diphosphate, adenosine diphosphate, deoxyadenosine diphosphate, deoxythymidine diphosphate, guanosine triphosphate, deoxycytidine triphosphate, uridine triphosphate, deoxythymidine triphosphate, adenosine triphosphate, and deoxyadenosine triphosphate reference standards.

[0032] Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatography system coupled with an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, USA); vortex mixer (IKA, Germany); centrifuge (Eppendorf, Germany); nitrogen blow-drying concentrator (Organomation, USA); solid phase extraction apparatus (Waters, USA).

[0033] 2. Experimental Methods

[0034] 2.1 Preparation of mixed standard solutions

[0035] To establish analytical methods and evaluate their performance, a mixed standard solution containing multiple representative phosphate-containing metabolites was precisely prepared for subsequent systematic evaluation of derivatization efficiency, chromatographic separation capability, and detection sensitivity. Approximately 1.0 mg of each phosphate-containing metabolite reference standard was accurately weighed, dissolved in 50% (v / v) methanol aqueous solution, and diluted to 1 mL to prepare a single-standard stock solution with a concentration of approximately 1 mg / mL, which was stored at -80°C. Immediately before use, an appropriate amount of each single-standard stock solution was diluted and mixed with 50% methanol aqueous solution to prepare a mixed standard working solution containing all 33 target analytes, with a concentration range of 0.1–10 µM.

[0036] 2.2ClXIMA derivatization reaction

[0037] The aim is to convert highly polar phosphate-containing metabolites into derivatives with specific mass spectrometry characteristics through a two-step reaction, thereby solving the problems of weak retention and difficult separation when directly analyzed.

[0038] (1) Preparation of reaction solution A: Accurately weigh 14.55 mg of EDC and dissolve it in 1 mL of acetonitrile to obtain a 14.55 mg / mL EDC solution. Accurately weigh 9 mg of imidazole and dissolve it in 1 mL of acetonitrile to obtain a 9 mg / mL imidazole solution. Mix equal volumes of the two solutions to obtain reaction solution A, which should be prepared fresh for use.

[0039] (2) Preparation of reaction solution B: Accurately weigh 10 mg of ClXIMA and dissolve it in 1 mL of DMSO / acetonitrile solution to obtain a 10 mg / mL ClXIMA solution. Measure 4 µL of 1-methylimidazole (density approximately 1.03 g / mL) and add it to 1 mL of acetonitrile, mix well to obtain an approximately 41.6 mM 1-methylimidazole solution. Mix an equal volume of ClXIMA solution with the 1-methylimidazole solution to obtain reaction solution B, which should be prepared fresh before use.

[0040] After being activated by EDC, the phosphate group reacts with imidazole to form an active phosphate ester intermediate, preparing for subsequent derivatization.

[0041] (3) Derivatization steps: Take 100 µL of the mixed standard working solution into a 1.5 mL centrifuge tube and dry it under nitrogen. Add 30 µL of DMSO to the dried residue and vortex for 30 seconds to dissolve it completely. Then add 15 µL of reaction solution A, vortex for 10 seconds, and react in a 40℃ metal bath for 30 minutes. After the reaction is complete, remove the centrifuge tube, immediately add 35 µL of reaction solution B, vortex for 10 seconds, and react again in a 40℃ metal bath for 2 hours. After the reaction is complete, remove the tube and cool it to room temperature. Centrifuge at 13500 rpm for 5 minutes, and transfer the supernatant to an LC-MS vial for analysis. At the same time, a blank derivatization control group without ClXIMA (using an equal volume of DMSO / acetonitrile solution instead of the ClXIMA solution in reaction solution B) is set up for comparison. By reacting ClXIMA with an active intermediate, a hydrophobic phenylisoxazole group and a chlorine atom tag with a characteristic isotopic distribution are covalently introduced into the target metabolite, thereby achieving the dual effect of enhancing chromatographic retention and providing mass spectrometry screening labels.

[0042] 2.3 Liquid Chromatography-Mass Spectrometry Analysis Conditions

[0043] A suitable analytical method for ClXIMA derivatives was established to achieve efficient separation and high-sensitivity detection. A phenylhexyl column and an ammonium bicarbonate-methanol gradient system were employed to fully utilize the interaction between the hydrophobic groups introduced by the derivatives and the stationary phase, achieving effective separation of components in the mixture, particularly for structurally similar isomers. Simultaneously, it was anticipated that clear quasi-molecular ion peaks and characteristic isotopic peak clusters of the derivatives could be obtained in positive ion full scan mode, providing high-quality primary mass spectra for subsequent data mining based on the chloride isotope distribution (~3:1).

[0044] Chromatographic conditions:

[0045] Column: Agilent ZORBAX RRHD Eclipse Plus Phenyl-Hexyl (2.1 × 100 mm, 1.8 µm). Column temperature: 35℃. Injection volume: 2 µL. Mobile phase: Phase A was 5 mM ammonium bicarbonate aqueous solution; Phase B was 5 mM ammonium bicarbonate in 90% methanol aqueous solution (v / v). Flow rate: 0.3 mL / min. Gradient elution program: 0–3.0 min, 92% A → 80% A; 3.0–6.5 min, 80% A → 67% A; 6.5–7.0 min, 67% A → 45% A; 7.0–9.0 min, 45% A → 5% A; 9.0–10.9 min, hold at 5% A; 10.9–11.0 min, 5% A → 92% A; then equilibrate at 92% A for 2 min. Total run time: 13 minutes.

[0046] Mass spectrometry conditions:

[0047] Ion source: Dual AJS ESI electrospray ionization source, positive ion mode. Drying gas temperature: 325℃. Drying gas flow rate: 11 L / min. Nebulizer pressure: 35 psig. Sheath gas temperature: 350℃. Sheath gas flow rate: 11 L / min. Capillary voltage: 4000 V. Nozzle voltage: 500 V. Scan mode: Full scan. Scan range: m / z 100 – 1000. Reference ions: m / z 121.050873 and 922.009798.

[0048] 3. Results and Analysis

[0049] 3.1 Optimization of chromatographic conditions

[0050] 3.1.1 Optimization of chromatographic columns

[0051] A comparative analysis was conducted using a standard ACQUITY HSS T3 column (1.8 μm, 100 mm × 2.1 mm) and an Agilent ZORBAXRRHD Eclipse Plus Phenyl-Hexyl (2.1 × 100 mm, 1.8 µm). For example... Figure 1 As shown, when using an HSS T3 column for separation, the derivatized mixed standards were eluted within 8-10 minutes, showing significant improvement in retention, but the peak shape was poor, with many extraneous peaks and severe peak overlap. When using a Phenyl-Hexyl column for separation, the retention of the mixed standards was improved, the peak shape was better, and baseline separation was achieved, greatly improving the resolution of the detected samples.

[0052] 3.1.2 Optimization of mobile phase additives

[0053] The addition of volatile ammonium salts can weaken the interaction between phosphate compounds and the active sites of the chromatographic column packing material through ion shielding, thereby effectively reducing peak tailing. Therefore, this study systematically compared the elution effects of ammonium bicarbonate, ammonium formate, and ammonium acetate on standards (e.g., Figure 2 (As shown). In the mobile phase with added ammonium formate and ammonium acetate, the peak shape of the target compound broadened, multiple standards showed shoulder peaks, and adenosine monophosphate exhibited an abnormal double peak phenomenon in the mobile phase with ammonium acetate as the additive. When ammonium bicarbonate was used as the mobile phase additive, the retention behavior of each component in the mixed standard was enhanced, the peak shape was symmetrical and sharp, and effective baseline separation was achieved.

[0054] 3.2 Improved chromatographic behavior

[0055] like Figure 3 As shown, the underivatized mixed standards exhibited extremely weak retention on the reversed-phase column, with most compounds eluting within 0.5–1.5 minutes, exhibiting broad peaks and severe overlap, making effective separation impossible. After ClXIMA derivatization, the retention times of all target metabolites were significantly prolonged, ranging from 1.5 to 9.5 minutes. The introduction of the hydrophobic phenylisoxazole group increased the hydrophobicity of the compounds and enhanced the π-π interaction with the phenyl stationary phase, resulting in better chromatographic retention and separation. This demonstrates that the ClXIMA derivatization strategy can significantly prolong the retention time of highly polar metabolites on reversed-phase chromatography and improve peak shape, providing a basis for solving co-elution problems.

[0056] 3.3 Isomer Separation Effect

[0057] Meanwhile, due to the strong hydrophilicity of phosphate groups and the similar polarity among metabolites, phosphate-containing compounds often form multiple pairs of structurally similar isomers, such as structural isomers, epimers, and positional isomers. These isomers regulate different physiological pathways within cells, making their effective chromatographic separation in LC-MS analysis both significant and challenging. Thanks to the excellent chromatographic separation capabilities of ClXIMA derivatization, three pairs of isomers (deoxyguanosine monophosphate and adenosine monophosphate, galactose-6-phosphate and mannose-6-phosphate, and glucose-1-phosphate and glucose-6-phosphate) were all well separated. Specifically, deoxyguanosine monophosphate and adenosine monophosphate produce the same parent ion, and before derivatization, the two compounds co-eluted in a reverse chromatographic system, affecting their accurate detection in real samples. However, after ClXIMA derivatization, the two compounds can be baseline separated. For the other two pairs of isomers, there are currently few studies on the chromatographic separation of galactose-6-phosphate and mannose-6-phosphate, and glucose-1-phosphate and glucose-6-phosphate, and the liquid chromatography method required to achieve complete separation is approximately 30 minutes. Through ClXIMA derivatization, these two pairs of isomers also achieved baseline separation in a shorter time (11 minutes elution method), significantly shortening the sample analysis time. Therefore, the ClXIMA derivatization method can significantly improve the separation efficiency of highly polar phosphate-containing compounds and enhance the detection capability of isomers. The method of this invention successfully achieved baseline separation of three pairs of difficult-to-separate isomers (such as...). Figure 4 (as shown)

[0058] dGMP and AMP: After derivatization, dGMP and AMP eluted in the chromatogram at 4.879 min and 6.034 min, respectively. The retention times of the two were significantly different, achieving baseline separation with a resolution (Rs) greater than 1.5.

[0059] Gal-6-P and Man-6-P: The retention times of these two hexose phosphate isomers after derivatization were 2.320 min and 1.752 min, respectively, achieving effective separation.

[0060] Glc-1-P and Glc-6-P: By derivatization and optimization of liquid phase separation conditions, the retention times of Glc-1-P and Glc-6-P were 4.256 min and 2.194 min, respectively, thus achieving complete separation of these two hexose phosphate isomers.

[0061] This method demonstrates superior separation capabilities for key, difficult-to-separate isomer pairs such as dGMP / AMP, Glc-1-P / Glc-6-P, and Gal-6-P / Man-6-P, which is difficult to achieve with existing methods.

[0062] 3.4 Mass Spectrometry Characteristics and Identification

[0063] Due to the introduction of chlorine by the derivatization reagent, the mass spectra of all ClXIMA derivatives exhibit a characteristic chlorine isotope distribution pattern in positive ion full scan mode: their [M+H]⁺ ion peaks (mainly from...) At a mass number increase of approximately 2 Da, the [M+2+H]⁺ isotope ion peaks (mainly from...) appear. The intensity ratio of the two peaks is approximately 3:1, and this characteristic mass spectrometric fingerprint provides crucial evidence for the structural confirmation of this series of derivatives. Taking the UMP-ClXIMA derivative as an example (e.g....), Figure 5 As shown in the figure, its quasi-molecular ion peak is [M+H]. + For m / z 515.0732 ( The calculated value is 515.0729), and its [M+2+H] + The isotope peak is m / z 517.0719, and the intensity ratio of the two is approximately 3.0:1. and The natural abundance ratio (approximately 3:1) is highly consistent. This characteristic peak can serve as a key basis for rapidly identifying ClXIMA derivatized products from complex mass spectrometry data. The characteristic [M]⁺ / [M+2]⁺ isotope peak clusters and their stable abundance ratios generated by chlorine atoms in the mass spectrometry of ClXIMA derivatives theoretically verify that they can serve as reliable and specific mass spectrometry tags for high-throughput screening.

[0064] Meanwhile, this feature tag can effectively mitigate false positives caused by intrasource fragmentation during data analysis. Taking the key nucleotide sugar metabolite ADP-glucose (ADP-Glc) as an example, it plays a crucial role in energy storage and metabolic balance as a direct precursor to starch and glycogen synthesis. Figure 6As shown, underivatively derivatized ADP-Glc readily fragments within the ion source, producing significant fragment ions (m / z 428.0393). The signal is consistent with the mass number of the quasi-molecular ion peak [M+H]+ (m / z 428.0367) of underivatized ADP. Due to their high polarity, both exhibit similar retention behavior in chromatography, leading to easy co-elution and severe interference with mass-to-charge ratio-based identification and quantification. By introducing a chlorine elemental tag through derivatization, the ADP-Glc derivatized product will carry a chlorine elemental tag, distinguishing it from the signal of fragment ions from the source fragments. This enables reliable identification of ADP-Glc in complex samples, significantly improving analytical accuracy.

[0065] Example 2: Screening for phosphate-containing metabolites in real biological samples using a Python data mining program based on characteristic isotope signals.

[0066] 1. Sample pretreatment and derivatization

[0067] This step applies the method of this invention to a real, complex biological matrix (mouse liver tissue) to verify its practical efficacy in non-targeted metabolomics analysis. The aim is to maximize the acquisition of endogenous phosphate-containing compounds in liver tissue by precipitating proteins with organic solvents and extracting small-molecule metabolites, while simultaneously removing interference from large molecules. A weak anion-exchange solid-phase extraction column is used to selectively enrich and purify negatively charged phosphate-containing metabolites, reducing sample matrix complexity and improving the relative concentration of target analytes and the signal-to-noise ratio. All potential phosphate-containing metabolites extracted from the biological sample are converted in batches into ClXIMA derivatives with characteristic chlorine isotope tags, similar to the processing of standards, laying a unified chemical foundation for subsequent high-throughput data mining.

[0068] Animal model: A cholestasis mouse model induced by α-naphthyl isothiocyanate (ANIT) was used. Liver tissue was collected from 6 mice in the model group and 6 mice in the control group, with each sample containing approximately 100 mg.

[0069] Sample extraction: Liver tissue was homogenized thoroughly with 200 µL of physiological saline in a grinding tube. 800 µL of methanol pre-cooled to -20°C was added, and the mixture was vortexed for 3 min. The mixture was then centrifuged at 13500 rpm for 10 min at 4°C, and the supernatant was collected. The residue was extracted once more with 400 µL of 80% cold methanol aqueous solution, and the supernatants from both extractions were combined.

[0070] Enrichment and purification: The combined supernatants were dried under nitrogen. The residue was redissolved in 100 µL of 50 mM ammonium acetate solution (pH 4.5). Enrichment was performed using a Waters Oasis WAX (30 mg / 1 cc) solid-phase extraction column. The column was activated sequentially with 200 µL of methanol and 200 µL of 50 mM ammonium acetate (pH 4.5). After loading the sample, the column was washed with 200 µL of 50 mM ammonium acetate (pH 4.5) and 200 µL of methanol. Finally, the target analyte was eluted with 200 µL of methanol solution containing 10% ammonia. The eluent was dried under nitrogen.

[0071] Derivatization: The dried sample was derivatized according to the steps in "2.2 ClXIMA derivatization reaction" in Example 1.

[0072] 2. LC-MS Data Acquisition

[0073] High-quality, high-resolution raw mass spectrometry data files were obtained. Optimized chromatographic-mass spectrometry conditions were used to capture, in a single analysis, the retention times, precise mass numbers, and complete isotopic peak shapes of all ClXIMA derivatives in the sample, providing comprehensive and reliable raw input for subsequent automated data mining. The derivatized sample was analyzed by LC-Q-TOF / MS full scan according to the "2.3 Liquid Chromatography-Mass Spectrometry Analysis Conditions" in Example 1.

[0074] 3. Data mining process based on Python

[0075] Develop and implement an automated and intelligent data processing strategy to rapidly and accurately locate target compounds from complex full-scan mass spectrometry data. Transform the proprietary-format raw data acquired by the instrument into a structured, machine-readable list of characteristic peaks, completing standardized steps such as peak detection and alignment, and preparing a standardized data pool for developing specific screening algorithms. Automated logical judgment based on chlorine isotope characteristics is executed to accurately locate potential targets conforming to the mass spectrometry patterns of ClXIMA derivatives from thousands of characteristic peaks. The core expectation is to achieve high-throughput and high-specificity preliminary screening, greatly reducing the workload of manual data browsing and minimizing subjective errors. Chemical identity is confirmed for the candidate peaks screened by the machine. Molecular formulas are derived through precise mass numbers, and combined with database retrieval and secondary mass spectrometry analysis, the screened characteristic signals are transformed into known or unknown metabolites with well-defined chemical structures, completing the identification from data to biological entities. By mapping differentially expressed metabolites to known metabolic pathways, the main biochemical pathways they perturb in specific pathological models are revealed.

[0076] 3.1 Data Preprocessing

[0077] The acquired raw mass spectrometry data files (.d) were converted to the open-source .abf format using the open-source software MSConvert (ProteoWizard). The .abf files were then imported into MS-DIAL software for non-targeted processing, with key parameters set as follows: mass tolerance 10 ppm, retention time tolerance 0.2 min, and minimum peak height 5000. The software automatically performed peak detection, denoising, alignment, and exported a table file containing the following information for each characteristic peak: peak number, retention time (min), m / z value, peak intensity, and its corresponding isotopic peak information.

[0078] 3.2 Screening ClXIMA derivatives using Python scripts

[0079] A Python script (key code logic is shown below) was written to automatically filter the exported characteristic peak table. Utilizing the characteristic isotope mass spectrometry signal of chlorine atoms, a Python-assisted data mining program was developed to rapidly extract potential phosphate-containing metabolites directly from LC-MS primary mass spectrometry data.

[0080] The preprocessing of the data using the Python-assisted data mining program is as follows: Data acquired via UHPLC-Q-TOF / MS in full scan mode is converted to .abf format using MSConvert software, and then imported into MS-DIAL software for automated peak acquisition and alignment. Subsequently, the processed data is output, and the exported data file contains peak intensity, retention time, m / z values, and isotope peak information.

[0081] The screening rules based on the Python-assisted data mining program are as follows: First, precursor ions containing both the M peak and the M+2 isotope peak are selected from the exported data files. Then, metabolites with a peak intensity ratio between 2.75 and 3.25 for the M peak and the M+2 isotope peak are selected as potential ClXIMA derivatives; other precursor ions are filtered out.

[0082] 3.3 Result Verification and Evaluation

[0083] The above Python script was used to analyze the LC-MS data of liver tissue samples from 6 cholestasis model mice. A total of 323 potential ClXIMA derivatization characteristic peaks that met the chlorine isotope characteristics (M / (M+2) intensity ratio between 2.75 and 3.25) were automatically screened from thousands of chromatographic peaks.

[0084] For these characteristic peaks, the "Molecular Formula Generator" function in Agilent MassHunter qualitative analysis software was used to calculate their possible molecular formulas based on their accurate mass numbers (typically with a mass error <5 ppm) and chlorine isotope fit. The ClXIMA residue portion was then subtracted from the derived derivative molecular formula. Considering the derivatization reaction that loses one molecule of water, the actual addition is... ), thus obtaining the molecular formula of the original metabolite.

[0085] The original molecular formula was compared with metabolite databases such as HMDB, METLIN, and KEGG. After preliminary matching, the structure was further verified by combining secondary mass spectrometry (MS / MS) fragment ion information. Ultimately, 194 endogenous metabolites were successfully identified, mainly including nucleotides and their derivatives, sugar phosphates, organophosphates, and glycerophosphates.

[0086] 3.4 Metabolic pathway analysis

[0087] The identified differentially metabolites were imported into the MetaboAnalyst 5.0 online platform for pathway analysis. The results showed that pathways such as purine metabolism, pyrimidine metabolism, glycolysis / gluconeogenesis, and the pentose phosphate pathway were significantly perturbed in the liver tissue of cholestasis model mice. In particular, the levels of energy metabolism-related molecules such as ATP, ADP, and AMP, as well as glucose-6-phosphate intermediates, were significantly altered, providing new data support for elucidating the metabolic reprogramming mechanism of cholestasis.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting phosphate-containing metabolites, characterized in that, Includes the following steps: The phosphate-containing metabolites in the test sample were derivatized using a chlorination reagent. The derivatized samples were analyzed by liquid chromatography-mass spectrometry to obtain mass spectrometry data. Based on the characteristic isotopic signal of chlorine in the chlorinated reagent from the mass spectrometry data, the derivatized phosphate-containing metabolites in the sample to be tested are identified.

2. The method according to claim 1, characterized in that, The identification based on characteristic isotope signals specifically includes: Precursor ions that simultaneously exhibit both a mass-to-charge ratio (M) peak and an isotopic (M+2) peak are screened from the mass spectrometry data; Calculate the peak intensity ratio of the M peak to the (M+2) peak; Precursor ions with peak intensity ratios within a preset range are identified as potential derivatized metabolites.

3. The method according to claim 2, characterized in that, The preset range is 2.75 to 3.

25.

4. The method according to claim 2, characterized in that, The filtering and calculation steps are implemented using a Python program.

5. The method according to claim 1, characterized in that, The derivatization treatment with the chlorinated reagent includes: The pretreated sample was mixed with a first reaction solution containing carbodiimide and imidazole to carry out the first reaction. Then, a second reaction solution containing a chlorinated reagent and methylimidazole is added to carry out the second reaction; The chlorination reagent is [5-(2-chlorophenyl)isoxazol-3-yl]methylamine.

6. The method according to claim 5, characterized in that, The conditions for the first reaction were: reacting at 40°C for 30 minutes; the conditions for the second reaction were: reacting at 40°C for 2 hours.

7. The method according to claim 1, characterized in that, Before the chlorination of the phosphate-containing metabolites, a sample pretreatment and enrichment step is also included, wherein the enrichment step is performed using a weak anion exchange solid-phase extraction column.

8. The method according to claim 7, characterized in that, The sample pretreatment and enrichment steps specifically include: Biological tissue samples were mixed with physiological saline and pre-cooled methanol, homogenized, centrifuged, and the supernatant was collected, combined, and concentrated to obtain the residue. The residue was redissolved in ammonium acetate buffer solution and extracted using an activated weak anion exchange solid phase extraction column. After rinsing with ammonium acetate buffer and methanol, the target compound was eluted with ammonia-methanol solution to obtain the enriched sample.

9. The method according to claim 1, characterized in that, In the aforementioned liquid chromatography-mass spectrometry analysis: The column packing material is phenyl-bonded silica gel; Mobile phase A is an aqueous solution of ammonium bicarbonate, and mobile phase B is an aqueous solution of ammonium bicarbonate in methanol, wherein the volume concentration of methanol is 90%. A gradient elution procedure was used.

10. The application of the method of any one of claims 1 to 9 in the rapid screening of phosphate-containing metabolites in non-targeted metabolomics analysis or in the screening or study of biomarkers associated with cholestatic diseases.

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