Bile acid high-throughput detection method based on ultra-high performance liquid chromatography-tandem mass spectrometry and application of bile acid high-throughput detection method

Abstract

The invention provides a bile acid high-throughput detection method based on ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof. The method comprises the following steps: pre-treating a sample to be detected, passing through a C18 solid-phase microextraction column, and collecting an eluent as a test solution; carrying out separation and detection by adopting ultra-high performance liquid chromatography-tandem mass spectrometry; mobile phases adopted by the ultra-high performance liquid chromatography comprise a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.095-0.105% formic acid aqueous solution containing 4.95-5.05 mM ammonium acetate, and the mobile phase B is an acetonitrile-methanol solution with the volume ratio of 3: (0.95-1.05); carrying out gradient elution; and establishing a linear regression equation of each bile acid subtype for quantitative calculation by mixing standard substances with different concentrations. The efficient extraction and detection method for the bile acid sample is established, separation and accurate quantification of 50 bile acid compounds are achieved, and the false positive problem caused by co-elution of isomeride bile acid in a traditional detection method is remarkably solved.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, specifically relating to a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry and its application. Background Technology

[0002] Bile acids, as core products of cholesterol metabolism, are not only key mediators in emulsifying fats, regulating fats and fat-soluble vitamins, and their transport and distribution, but also signaling molecules involved in the regulation of glucose and lipid metabolism through receptor pathways such as FXR and TGR5, possessing important physiological functions. As major components of bile, the subtype composition and dynamic balance of bile acids are directly related to enterohepatic circulation function, exhibiting specific changes in pathological processes such as non-alcoholic fatty liver disease, intracranial cholestasis of pregnancy (ICP), and inflammatory bowel disease. In medical research, accurate qualitative and quantitative analysis of bile acids is of practical significance for related disease research, liver function assessment, and the evaluation of drug metabolism and treatment efficacy. Therefore, accurate detection of bile acid components is extremely important in medical research and disease diagnosis related to liver and digestive metabolism. Bile acids can be classified into two types according to their structure: free and bound. Free bile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. Conjugated bile acids are products of the conjugation of free bile acids with glycine or taurine, mainly including glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid. Bile acids can be classified into primary, secondary, and tertiary bile acids according to their origin. Primary bile acids are synthesized directly from cholesterol in hepatocytes, including cholic acid, chenodeoxycholic acid, and their conjugation products with glycine or taurine. Secondary bile acids are produced in the intestine by bacterial action, where the 7α-hydroxyl group of primary bile acids is deoxygenated, mainly including deoxycholic acid and lithocholic acid, and their conjugation products with glycine or taurine. Tertiary bile acids are metabolic products of reabsorbed secondary bile acids in the liver and intestines, including sulfolithocholic acid and ursodeoxycholic acid.

[0003] Due to limitations in detection techniques, current clinical testing primarily focuses on total bile acids or the analysis of a limited number of bile acid species (usually <30). Detection of bile acids using kit-based enzymatic cycling and high-performance liquid chromatography (HPLC) suffers from poor reliability in both quantitative and qualitative analysis. Furthermore, the sample pretreatment involves complex steps such as derivatization, requiring precise control over reaction temperature and reagent quality. This results in low throughput, relatively poor sample reproducibility, and high experimental costs, significantly limiting the clinical applications of these two methods. Additionally, bile acid detection often involves biological samples such as plasma, intestinal contents, and liver tissue, which have complex matrices. Conventional extraction methods often result in extracts containing inorganic salts and other impurities, negatively impacting baseline noise, compound response values, and method stability, while also causing significant damage to the chromatographic column and ion source nozzle. Passing sample extracts through a C18 packed solid-phase microextraction column is a stable and efficient method for purifying samples and enriching target compounds. It offers good recovery rates for weakly polar substances, effectively removes hydrophilic salts and other impurities, and, when used with a matching solid-phase microextraction device, enables high-throughput sample processing. Compared to traditional methods, it does not add extra processing time or alter compound structures, thus improving data quality while maintaining experimental efficiency. It ensures timeliness for large-scale sample extraction and avoids degradation of bile acids during pretreatment.

[0004] Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) offers advantages such as high qualitative accuracy, high sensitivity, minimal interference from impurities, and simple pretreatment, effectively improving detection throughput and method accuracy, and is widely used in medical and scientific research. However, bile acids have relatively simple chemical core structures, and the differences between various subtypes of secondary bile acids often lie only in their spatial structure. Isomers or isotopes (such as 3-β-cholic acid and ω-mouse cholic acid, GCDCA / GDCA / GUDCA, etc.) have the same nucleus-mass ratio and similar chemical structures, making them difficult to separate effectively in conventional detection systems. This leads to insufficient detection specificity and a tendency for false positives or inaccurate concentration readings. Currently reported traditional methods and existing patents have the following drawbacks: (1) Insufficient chromatographic separation: Under gradient conditions of using a conventional C18 column and a general-purpose pure acetonitrile or methanol as the organic phase, the separation of structurally similar isomers of bile acids (such as cholic acid CA / isocholic acid ICA, glycochenodeoxycholic acid GCDCA / glycoursodeoxycholic acid GUDCA) is poor, resulting in the target peak not being effectively separated during mass spectrometry detection, and ion interference affecting the detection accuracy. (2) Poor mobile phase compatibility: It is difficult to balance the elution efficiency of hydrophobic bile acids (such as lithocholic acid LCA) and conjugated bile acids (such as taurocholic acid TCA) using methanol-water as the mobile phase system, resulting in unsatisfactory peak response values ​​and peak shapes of compounds in the detection results. (3) Simple gradient program: Simple linear gradients cannot be adapted to the elution separation of complex bile acid substances, and isotopic substances (such as deoxycholic acid DCA and porcine deoxycholic acid HDCA with m / z 407.3) are easily unable to be separated due to co-elution factors.

[0005] CN108072704B discloses a method for detecting bile acids in feces based on liquid chromatography-mass spectrometry (LC-MS), which uses a C8 column and an acetonitrile / water mobile phase system. It separates only 22 bile acids and does not mention the separation of related isomers. CN110596295A discloses a method for detecting bile acids that uses a conventional C18 column. Isomer separation relies on extended detection time (>30 min) and does not completely resolve interference between isomers.

[0006] Given the shortcomings of existing literature or patents regarding bile acid detection methods, and the increasing demand for precise qualitative and quantitative analysis of different bile acid subtypes in modern medical testing, metabolism, and drug development research, which goes beyond the detection of total bile acids, it is of great practical significance to establish a high-throughput method for qualitative and quantitative analysis of bile acid compounds. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and its applications. This detection method, based on UPLC-MS / MS technology, can detect 50 bile acid compounds, overcoming the limitations of existing technologies in terms of the limited range of bile acid detections and the difficulty in isomer separation. Furthermore, the pretreatment method of this invention is relatively simple, avoiding complex derivatization and other processing steps, thus achieving a high-throughput, high-stability, and high-accuracy detection method for bile acid compounds. This method has high commercial value and broad application prospects in the field of medical research and detection.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry, the method comprising: (1) Pre-treat the sample to be tested, then pass it through a C18 solid phase microextraction column, and collect the eluent as the test solution; (2) The test solution and the standard mixed solution are separated and detected by ultra-high performance liquid chromatography-tandem mass spectrometry; the mobile phase used in the ultra-high performance liquid chromatography includes: mobile phase A: 0.095-0.105% (e.g., 0.095%, 0.1%, or 0.105%) aqueous formic acid containing 4.95-5.05 mM (e.g., 4.95 mM, 5.00 mM, or 5.05 mM, etc.) ammonium acetate; mobile phase B: acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 3:(0.95-1.05) (e.g., 3:0.95, 3:1, or 3:1.05, etc.); gradient elution is performed using the mobile phase, and the gradient elution procedure is as follows: From 0 to 0.5 min, the volume fraction of mobile phase A is 90-90.5%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90-90.5% to 78-78.5%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78-78.5% to 68-68.5%, with the remainder being mobile phase B. During the 2.5-3.5 min interval, the volume fraction of mobile phase A was 68-68.5%, with the remainder being mobile phase B. Between 3.5 and 5.5 minutes, the volume fraction of mobile phase A changes uniformly from 68-68.5% to 64.5-65%, with the remainder being mobile phase B. From 5.5 to 9 minutes, the volume fraction of mobile phase A changed uniformly from 64.5-65% to 58-58.5%, with the remainder being mobile phase B; During the 9th to 10th minute, the volume fraction of mobile phase A changed uniformly from 58-58.5% to 57-57.5%, with the remainder being mobile phase B; Between 10 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 57-57.5% to 33-33.5%, with the remainder being mobile phase B. From 18 to 19.2 min, the volume fraction of mobile phase A changed uniformly from 33-33.5% to 5-5.5%, with the remainder being mobile phase B; From 19.2 to 20.3 min, the volume fraction of mobile phase A was 5-5.5%, with the remainder being mobile phase B; Between 20.3 and 20.4 minutes, the volume fraction of mobile phase A changed uniformly from 5-5.5% to 90-90.5%, with the remainder being mobile phase B. From 20.4 to 23 min, the volume fraction of mobile phase A was 90-90.5%, with the remainder being mobile phase B; (3) Linear regression equations for each bile acid subtype compound were established by mixing standard samples of different concentrations for quantitative calculation.

[0009] The detection method of the present invention can simultaneously meet the requirements of relatively simplified pretreatment, strong qualitative and quantitative capabilities, ideal separation effect of isomer bile acids, and relatively comprehensive coverage of bile acid subtypes.

[0010] This invention provides a detection method applicable to the determination of 50 bile acids in biological samples related to bile acid metabolism (plasma, liver, intestinal contents, and feces). It establishes a mass spectrometry (MRM) acquisition method using standards for each bile acid subtype, optimizes the chromatographic column and mobile phase formulations, and simultaneously optimizes the gradient method to achieve separation of isomers of bile acids. For example, this invention can effectively distinguish between isolithocholic acid and isochocholic acid, which is completely indistinguishable in conventional detection methods. These substances are often defined as the same substance in conventional detection, affecting the accuracy of both qualitative and quantitative results. Therefore, the optimized method of this invention significantly improves the reliability of bile acid detection. This invention controls the detection gradient time to 23 minutes, compared to similar methods which generally exceed 30 minutes, effectively increasing throughput and reducing costs.

[0011] Preferably, in step (1), the sample pretreatment step includes: The collected sample was mixed with the extract, frozen with dry ice, and then shaken to promote sample lysis. The sample was extracted by ultrasonic extraction in an ice-water bath, frozen with dry ice, and then centrifuged to collect the supernatant. The supernatant was filtered, and the filtrate was collected. The filtrate was mixed with water and passed through a C18 solid-phase microextraction column. After rinsing with pure water, the sample was eluted with pure methanol at least three times. The eluent was collected as the test solution.

[0012] Preferably, the extract is an acetonitrile-methanol-water solution, wherein the volume ratio of acetonitrile, methanol, and water is (1.9-2.1):(1.9-2.1):(0.9-1.1). Here, "1.9-2.1" can be, for example, 1.9, 2, or 2.1; and "0.9-1.1" can be, for example, 0.9, 1, or 1.1.

[0013] Preferably, the dry ice freezing time is 4-6 minutes, for example, 4 minutes, 5 minutes or 6 minutes.

[0014] Preferably, the oscillation conditions are: oscillation at 1200-1250 rpm (e.g., 1200 rpm, 1220 rpm, 1240 rpm, or 1250 rpm, etc.) for 10-15 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes, etc.

[0015] Preferably, the ultrasonic extraction step takes 8-12 minutes, for example, 8 minutes, 9 minutes, 10 minutes, 11 minutes or 12 minutes.

[0016] Preferably, the centrifugation step is performed at a rotation speed of 11,000-12,000 rpm (e.g., 11,000 rpm, 11,500 rpm, or 12,000 rpm) and a temperature of 0-4℃ (e.g., 0℃, 1℃, 2℃, 3℃, or 4℃) for 8-12 minutes, such as 8 minutes, 9 minutes, 10 minutes, 11 minutes, or 12 minutes.

[0017] Preferably, the filtration uses a disposable needle filter with a 0.22 μm nylon 66 membrane.

[0018] Preferably, the volume ratio of the filtrate to water is 1:(3.5-4.5), for example, it can be 1:3.5, 1:4 or 1:4.5, etc.

[0019] Preferably, the packing material of the C18 solid-phase microextraction column is Sep-Pak C18 1 cc Vac Cartridge.

[0020] This invention employs solid-phase microextraction (SPE) column purification during pretreatment, avoiding time-consuming and costly methods such as derivatization, concentration, and drying. This helps maintain the original state of bile acids and prevents degradation, while also removing background impurities, effectively improving quantitative accuracy and efficiency. This invention optimizes the composition of the extract to ensure extraction efficiency, while adding a purification step using a C18 packed solid-phase microextraction column. It also optimizes the elution method and eluent volume, which helps enrich bile acids and remove impurities, resulting in fewer background peaks, a lower baseline, and improved peak shape in the sample. Furthermore, it verifies that the optimized method effectively improves the extraction recovery rate, thereby significantly improving the accuracy of qualitative and quantitative analysis without affecting the pretreatment throughput.

[0021] Preferably, in step (2), the ultra-high performance liquid chromatography column is a Waters ACQUITY UPLC BEHC18 column, with a size of 1.7 μm and 2.1 mm × 100 mm; the column temperature is 34-36℃, for example, 34℃, 35℃ or 36℃.

[0022] Preferably, in step (2), the ultra-high performance liquid chromatograph is an ACQUITY UPLC I-Class PLUS, Waters, and the injection volume is 1.8-2.2 μL, for example, 1.8 μL, 2 μL or 2.2 μL.

[0023] Preferably, in step (2), the mass spectrometry conditions are as follows: an electrospray ionization (ESI) source is used, and negative ion mode is used for detection. The instrument parameters are as follows: curtain gas is 35 psi, ion spray voltage is -4500V, ion source temperature is 550℃, nebulizing gas is 50 psi, and auxiliary gas is 55 psi.

[0024] In this invention, a Sciex Qtrap 6500+ triple quadrupole mass spectrometer was used to detect 50 bile acids. The triple quadrupole mass spectrometer adopted the MRM detection mode. The parent ion / daughter ion detection ion pairs of the analytes are shown in Table 1 below; the declustering voltage and collision energy parameters of the analytes are shown in Table 2 below.

[0025] Table 1 Table 2 Preferably, in step (2), the standard mixed solution comprises: dehydrolithocholic acid, isolithocholic acid, lithocholic acid, 2,3-demethyldeoxycholic acid, 7-ketolithocholic acid, 12-ketolithocholic acid, protocholic acid, ursodeoxycholic acid, porcine deoxycholic acid, chenodeoxycholic acid, deoxycholic acid, isodeoxycholic acid, dehydrocholic acid, 7,12-diketolithocholic acid, 7-ketodeoxycholic acid, 12-dehydrocholic acid, 3-dehydrocholic acid, ursolic acid, α-mouse cholic acid, β-mouse cholic acid, porcine cholic acid, allocholic acid, cholic acid, glycinelithocholic acid, and glycineursodeoxycholic acid. Glycine deoxycholic acid, glycodeoxycholic acid, glycodehydrocholic acid, glycocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, isolithocholic acid, isurolectocholic acid, isosucrase deoxycholic acid, 3β-deoxycholic acid, norcholic acid, 3β-cholic acid, ω-mouse cholic acid, glycochenodeoxycholic acid, lithocholic acid sulfate, glycocaine, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, and chenodeoxycholic acid-3-β-glucuronic acid.

[0026] Preferably, the concentration range of each bile acid subtype standard in the standard mixed solution is 0.1-5000 ng / mL, for example, it can be 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 2000 ng / mL, 4000 ng / mL or 5000 ng / mL, etc.

[0027] Secondly, the present invention provides the application of the high-throughput bile acid detection method based on ultra-high performance liquid chromatography-tandem mass spectrometry described in the first aspect in the detection of bile acids.

[0028] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0029] Compared with the prior art, the present invention has the following beneficial effects: (1) First, this invention develops a method for acquiring precursor ion-daughter ion pairs of various bile acid subtypes using a mass spectrometry MRM acquisition method based on Qtrap6500+. Since most precursor ions of bile acids are difficult to break down, a precursor ion-precursor ion acquisition channel is needed for data acquisition. Furthermore, due to the similarity in structure among bile acids, 40 bile acid subtypes exhibit isomerism, with overlapping molecular weights (see Table 3 below). Therefore, liquid chromatography is required to improve the elution and separation of each substance to achieve reliable qualitative and quantitative analysis. This invention further optimizes the mobile phase formulation and gradient method, and selects the optimal chromatographic column to achieve complete separation of 50 bile acid subtypes within a shorter detection time.

[0030] Table 3 (2) Most bile acid compounds have isomers with the same molecular weight. Due to the limitations of the triple quadrupole mass spectrometry qualitative and quantitative principle, targeted quantification requires the separation of chromatographic peaks of compounds with the same mass-to-charge ratio. Co-elution can lead to false positives in qualitative analysis and inaccurate quantitative results. The detection method of this invention optimizes the mobile phase formulation and liquid phase elution gradient to fully separate each compound, achieving separation of isomer bile acid chromatographic peaks and avoiding concentrated peaks of compounds in the middle of the gradient, effectively reducing the probability of false positives and ensuring detection accuracy. To achieve the above elution method, this invention preferably uses the WatersACQUITY UPLC BEH C18 column (1.7 μm 2.1×100 mm). The trifunctional bonded BEH (ethylene bridged hybrid) particles have a wider usable pH range (pH 1-12) and excellent low pH stability. Compared with traditional C18 columns, it has the characteristics of high column efficiency and high separation efficiency, and its performance in bile acid projects is significantly better than other chromatographic columns. Meanwhile, in traditional methods that use pure acetonitrile as the organic phase, multiple isomers of bile acids cannot be distinguished. This invention innovatively optimizes the organic phase portion of the mobile phase, using an acetonitrile:methanol ratio of 3:1 as the organic phase collection method. At the same time, the elution gradient is optimized multiple times to achieve a liquid-phase method that can effectively separate various compounds in a shorter time.

[0031] (3) The optimized pretreatment method of this invention improves upon traditional methods by reducing concentration and derivatization, which affect throughput. It also prevents degradation or content changes in bile acids during extraction, increasing throughput while ensuring the accuracy of each subtype of bile acid. This invention uses an extraction solution formulation that, compared to the pure methanol or methanol-water extraction solutions reported previously, contains a portion of acetonitrile for more comprehensive extraction of bile acids, resulting in better recovery and increased detection quantity and efficiency. Furthermore, a C18 column impurity removal method is added. Due to the low retention of hydrophilic phases by C18 packing material, it effectively removes impurities such as salts, reduces sample background peaks, effectively improves detection recovery, and reduces matrix effects. Simultaneously, the solid-phase microextraction column method does not affect the structural changes of bile acids, is relatively simple to process, and increases pretreatment extraction throughput while avoiding distortion of detection results due to compound transformation.

[0032] (4) The solid phase microextraction column with C18 packing (the packing is Sep-Pak C18 1 cc VacCartridge) used in this invention can remove impurities from the sample while ensuring the recovery rate of bile acids. Under optimal conditions, the recovery rate of each bile acid in the test results ranges from 93.43% to 108.49%. Attached Figure Description

[0033] Figure 1 This is a flowchart of a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry.

[0034] Figure 2 To collect the extracted ion chromatogram of bile acid mixed standards.

[0035] Figure 3 Extraction ion flow chromatogram of the loading solution for collecting intestinal contents samples.

[0036] Figure 4 This is a comparison of ion chromatograms of the sample obtained by solid-phase microextraction column processing (dehydrolithocholic acid) using the pretreatment method of this invention.

[0037] Figure 5 This is a comparison of the ion chromatograms of the sample obtained by the pretreatment method of the present invention through solid-phase microextraction column (23-demethyldeoxycholic acid).

[0038] Figure 6 This is a comparison of ion chromatograms of the sample obtained by solid-phase microextraction column processing (porcine deoxycholic acid) using the pretreatment method of this invention.

[0039] Figure 7 This is a comparison of ion chromatograms (cholic acid) of the sample obtained by solid-phase microextraction column processing in the pretreatment method of this invention.

[0040] Figure 8Comparison of ion chromatograms of sample feed solution obtained by conventional processing methods (dehydrolithocholic acid).

[0041] Figure 9 Comparison of ion chromatograms of sample loading solution obtained by conventional processing methods (23-demethyldeoxycholic acid).

[0042] Figure 10 Comparison of ion chromatograms of sample loading solution obtained by conventional processing methods (porcine deoxycholic acid).

[0043] Figure 11 Comparison of ion chromatograms (cholic acid) obtained from sample loading using conventional processing methods.

[0044] Figure 12 The extracted ion chromatograms are shown for isomers (M / Z=375, 391, 401, 405, 514) in a bile acid mixed standard under a chromatographic column (T3 column) and a mobile phase system (phase A: 0.1% formic acid (5mM ammonium acetate) water, phase B: acetonitrile: methanol = 3:1).

[0045] Figure 13 The extracted ion chromatograms are shown for isomers (M / Z=375, 391, 401, 405, 514) in a bile acid standard under a chromatographic column (BEH column) and a mobile phase system (phase A: 0.01% formic acid water, phase B: pure acetonitrile).

[0046] Figure 14 The extracted ion chromatograms are shown for isomers (M / Z = 375, 391, 401, 405, 514) in a bile acid standard under a chromatographic column (BEH column) and a mobile phase system (phase A: 0.1% formic acid (5mM ammonium acetate) and water, phase B: pure acetonitrile).

[0047] Figure 15 The extracted ion chromatograms are shown for isomers (M / Z=375, 391, 401, 405, 514) in a bile acid mixed standard under a chromatographic column (BEH column) and a mobile phase system (phase A: 0.1% formic acid (5mM ammonium acetate) water, phase B: B acetonitrile: methanol = 3:1).

[0048] Figure 16 The extracted ion chromatograms are shown for isomers (M / Z = 389, 448, 464, 498) in a bile acid mixed standard under a chromatographic column (BEH column) and a mobile phase system (phase A: 0.1% formic acid (5mM ammonium acetate) water, phase B: B acetonitrile: methanol = 3:1). Detailed Implementation

[0049] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0051] Figure 1 This is a flowchart of a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). This invention uses 50 bile acid standards to establish a high-throughput detection method for bile acids based on an UPLC-MS / MS platform. Using mouse intestinal contents as an example, bile acid detection is performed. The main process is shown below.

[0052] Stock solutions of various bile acid standards were prepared, diluted to appropriate concentrations, and injected using a syringe pump. Mass spectrometry parameters, including the mass-to-nucleus ratio of the precursor and daughter ions, declustering voltage, and collision energy, were developed and optimized using a Qtrap6500+ mass spectrometer. The integrated parameters yielded the mass spectrometry method information. Since each subtype of bile acids involves multiple isomers, and the ion pairs obtained by mass spectrometry fragmentation in MRM mode are not significantly different, mass spectrometry alone cannot distinguish between the various bile acid subtypes. Qualitative analysis requires the assistance of an ultra-high performance liquid chromatography (UHPLC) system, relying on elution separation capabilities to separate the subtypes for accurate identification. This part involves the establishment and optimization process of the UHPLC method, including determining the mobile phase ratio and additive formulation, optimizing the chromatographic column, and optimizing the liquid phase elution gradient to achieve the separation of various bile acid compounds, thus establishing the optimal liquid phase method.

[0053] Example 1 This embodiment provides a high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry.

[0054] 1. Sample pretreatment.

[0055] Weigh an appropriate amount of sample (30 mg ± 1 mg) into a 2.0 mL centrifuge tube. If the intestinal contents sample has a high water content, it needs to be freeze-dried and weighed first. Add clean grinding beads to the centrifuge tube and 1000 μL of extraction buffer (V acetonitrile:V methanol:V water = 2:2:1). Seal the centrifuge tube tightly and vortex for 30 s to mix. Freeze the sample on dry ice for 5 minutes, then place the centrifuge tube containing the sample into a shaker and shake at 1200 rpm for 12 min to promote sample lysis. Extract by sonication in an ice-water bath for 10 minutes, then freeze again on dry ice for 5 minutes. Place the extracted sample symmetrically into a centrifuge and centrifuge at 12000 rpm and 4℃ for 15 min to remove impurities. Filter the supernatant through a 0.22 μm nylon 66 disposable syringe filter. Take 800 μL of the filtrate into a 5 mL centrifuge tube, add 3200 μL of pure water, and mix well to obtain the sample solution. Activate the C18 solid-phase microextraction column (Sep-Pak C18 1 cc Vac Cartridge) with 5 mL of methanol, wash the activated solid-phase microextraction column with 5 mL of pure water, load the pretreated sample solution obtained earlier, rinse with 1 mL of pure water, elute 3 times with 0.5 mL of pure methanol and collect and combine the eluents, then make up to 2 mL with pure methanol for instrument detection.

[0056] Standard preparation.

[0057] The standard concentration was 50 μg / mL, containing all bile acids listed in Table 1. Different concentrations of mixed standards were prepared by diluting with 50% acetonitrile-water for linear preparation, ranging from 0.1 ng / mL to 5000 ng / mL. Specific preparation methods are as follows: Preparation of Stock Solution A: Weigh approximately 5 mg of the standard into a 1 mL volumetric flask, and dilute to 1 mL with 50% acetonitrile aqueous solution to prepare a single-standard stock solution with a concentration of approximately 5 mg / mL. Store at -80℃.

[0058] Preparation of Stock Solution B: Transfer 100 μL of each single-standard stock solution A into a 10 mL volumetric flask, and dilute to volume with 50% acetonitrile water. The concentration of each bile acid standard is 50 μg / mL. After aliquoting, store at -20℃.

[0059] Preparation of stock solution C (prepare immediately before use): Transfer 40 μL of stock solution B into a 2 mL centrifuge tube and dilute to 1 mL with 50% acetonitrile water, resulting in a concentration of 2000 ng / mL.

[0060] Preparation of external standard stock solution D (prepare immediately before use): Transfer 100 μL of stock solution C to a 2 mL centrifuge tube and dilute to 1 mL with 50% acetonitrile water, resulting in a concentration of 200 ng / mL.

[0061] Preparation of external standard stock solution E (prepare immediately before use): Transfer 50 μL of stock solution D into a 2 mL centrifuge tube and dilute to 1 mL with 50% acetonitrile water, resulting in a concentration of 10 ng / mL.

[0062] Preparation of linear solutions for standard curves.

[0063] The gradients of the linear solutions for the standard curve are as follows: 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 2000 ng / mL, 4000 ng / mL, and 5000 ng / mL. Prepare according to the method shown in Table 4.

[0064] Table 4 Taking the preparation of LINE1 as an example: Transfer 10 μL of the standard curve stock solution E into a 1 mL liquid chromatography vial, and dilute to 1 mL with 50% acetonitrile water, resulting in a concentration of 0.1 ng / mL. Prepare the remaining linear solutions using the same method.

[0065] 2. Sample separation by liquid chromatography.

[0066] (1) The sample to be analyzed was loaded and separated using UHPLC, and 50 bile acid compounds were separated by chromatographic column.

[0067] The UHPLC model was ACQUITY UPLC I-Class PLUS (Waters), with an injection volume of 2 μL. The column model was ACQUITY UPLC BEH C18 (1.7 μm, 2.1 mm × 100 mm), and the column temperature was 35℃. The mobile phase was phase A: 0.1% formic acid aqueous solution (containing 5 mM ammonium acetate), and phase B: acetonitrile:methanol = 3:1. The flow rate was 0.4 mL / min. The mobile phase gradient was: From 0 to 0.5 min, the volume fraction of mobile phase A is 90%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90% to 78%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78% to 68%, with the remainder being mobile phase B. From 2.5 to 3.5 min, the volume fraction of mobile phase A was 68%, and the remainder was mobile phase B; Between 3.5 and 5.5 minutes, the volume fraction of mobile phase A changed uniformly from 68% to 64.5%, with the remainder being mobile phase B. Between 5.5 and 9 minutes, the volume fraction of mobile phase A changed uniformly from 64.5% to 58%, with the remainder being mobile phase B. Between 9 and 10 minutes, the volume fraction of mobile phase A changed uniformly from 58% to 57%, with the remainder being mobile phase B. Between 10 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 57% to 33%, with the remainder being mobile phase B. Between 18 and 19.2 minutes, the volume fraction of mobile phase A changed uniformly from 33% to 5%, with the remainder being mobile phase B. From 19.2 to 20.3 min, the volume fraction of mobile phase A was 5%, and the remainder was mobile phase B; Between 20.3 and 20.4 minutes, the volume fraction of mobile phase A changed uniformly from 5% to 90% A, with the remainder being mobile phase B. From 20.4 to 23 min, the volume fraction of mobile phase A was 90%, with the remainder being mobile phase B.

[0068] (2) Mass spectrometry detection: Fifty bile acids were detected using a Sciex Qtrap 6500+ triple quadrupole mass spectrometer. The external standard method was used to establish linear equations for peak area and concentration for each of the 50 bile acid compounds, and further qualitative and quantitative analysis of each bile acid compound in the sample was performed.

[0069] The triple quadrupole mass spectrometer uses an electrospray ionization (ESI) source and negative ion mode for detection. The parameters are as follows: curtain gas 35 psi, ion spray voltage -4500V, ion source temperature 550℃, nebulizer gas 50 psi, and auxiliary gas 55 psi.

[0070] After collecting data using the above liquid chromatography-mass spectrometry (LC-MS) method parameters, linear regression equations for each bile acid subtype were established using mixed standards of different concentrations for quantitative calculation. Retention times confirmed by the MRM method channel and individual standards were used for qualitative analysis of each bile acid subtype, thus establishing a qualitative and quantitative method for bile acids. Simultaneous detection of the sample loading solution confirmed the content of each bile acid subtype in the sample. The retention times of each substance are shown in Table 5. Table 5 The extracted ion chromatograms of the mixed bile acid standards collected using the above method are shown below. Figure 2 Ion chromatograms of intestinal contents samples are shown below. Figure 3The ion chromatograms extracted from the standard samples show that the peaks of each bile acid subtype in the sample are well-formed, without obvious tailing, and the baseline is flat. After sample collection, each bile acid subtype can be clearly and quantitatively detected, and there is no obvious background peak noise interference. The preferred method of this invention is feasible for practical application.

[0071] Example 2 This embodiment uses mouse plasma as an example to investigate the pretreatment method for bile acid sample extraction.

[0072] 1. Sample extraction.

[0073] Transfer 100 μL of plasma sample to a 2.0 mL centrifuge tube, add 900 μL of extraction buffer (V acetonitrile:V methanol:V water = 2:2:1); seal the centrifuge tube tightly and vortex for 30 s to mix. Freeze the sample tube on dry ice for 5 minutes, then sonicate it in an ice-water bath for 5 minutes. Place the extracted sample symmetrically into a centrifuge and centrifuge at 12000 rpm and 4℃ for 15 min to remove impurities. Filter the supernatant through a 0.22 μm nylon 66 disposable syringe filter for later use.

[0074] 2. Prepare the sample for column loading.

[0075] Option 1: Take 800 μL of filtrate into a 5 mL centrifuge tube, add 2200 μL of pure water and mix well to obtain the pretreatment sample solution.

[0076] Option 2: Take 800 μL of filtrate into a 5 mL centrifuge tube, add 3200 μL of pure water and mix well to obtain the pretreatment sample solution.

[0077] Option 3: Take 800 μL of filtrate into a 5 mL centrifuge tube, add 4200 μL of pure water and mix well to obtain the pretreatment sample solution.

[0078] 3. Pass through a C18 solid-phase microextraction column.

[0079] Column chromatography protocol 1: Activate a C18 solid-phase microextraction column (Sep-Pak C18 1 cc VacCartridge or Sep-Pak C18 3 cc Vac Cartridge) with 5 mL of methanol. Wash the activated solid-phase microextraction column with 5 mL of pure water. Load the pretreated sample solution obtained earlier, rinse with 1 mL of pure water, and then elute 3 times with 0.5 mL of pure methanol. Collect and combine the eluents, and then make up to 2 mL with pure methanol for analysis.

[0080] Column chromatography protocol 2: Activate a C18 solid-phase microextraction column (Sep-Pak C18 1 cc VacCartridge or Sep-Pak C18 3 cc Vac Cartridge) with 5 mL of methanol. Wash the activated solid-phase microextraction column with 5 mL of pure water. Load the pretreated sample solution obtained earlier, rinse with 1 mL of pure water, and then elute 3 times with 0.5 mL of pure methanol. Collect and combine the eluents, and then make up to 2 mL with pure methanol for analysis.

[0081] 4. Experimental results.

[0082] (1) To optimize the organic phase ratio concentration in the column-loaded samples, the optimal loading solution system was selected. Three ratios were compared: 800 μL sample supernatant + 2200 μL water, 800 μL sample supernatant + 3200 μL water, and 800 μL sample supernatant + 4200 μL water. The loading solution was prepared using the procedure in Scheme 1, and the concentrations of each bile acid were measured. The results of the organic phase ratio concentration optimization experiment are shown in Table 6 below.

[0083] Table 6 Experimental results show that using an 800 μL sample supernatant + 3200 μL water system as the column loading solution is optimal, maximizing sample recovery with minimal baseline background peaks, achieving recoveries ranging from 93.43% to 108.49%. The recoveries with an 800 μL sample supernatant + 2200 μL water system ranged from 88.22% to 140.15%, and those with an 800 μL sample supernatant + 4200 μL water system ranged from 70.65% to 146.75%.

[0084] (2) Optimization of solid-phase microextraction (SPE) columns with different packing volume specifications: Using an optimal ratio of 800 μL sample supernatant + 3200 μL water as the loading solution, SPE columns with different packing volume specifications were used: Sep-Pak C18 1 cc Vac Cartridge and Sep-Pak C18 3 cc Vac Cartridge. Each column was washed with 1 mL of pure water, followed by elution three times with 0.5 mL of pure methanol. The eluents were collected and combined, then diluted to 2 mL with pure methanol before being analyzed. The bile acid recovery rates obtained from different SPE column specifications were measured. The optimization results of SPE columns with different packing volume specifications are shown in Table 7 below.

[0085] Table 7 When using the Sep-Pak C18 3 cc Vac Cartridge, the recovery rate was 77.48%-107.32%, lower than the 93.43%-108.49% recovery rate under the preferred Sep-Pak C18 1 cc Vac Cartridge condition of this invention. Experimental results show that although the packing material formulation of the Sep-Pak C18 3 cc Vac Cartridge solid-phase microextraction column is consistent with the preferred solid-phase microextraction column of this invention, the larger packing material itself leads to incomplete adsorption or elution of substances. The recovery rate of bile acids after passing through is worse than that of the 1 cc specification, indicating that excessively large packing volume is less suitable for this experimental method, failing to utilize the material retention capacity of large-sized packing material and instead causing more material loss. Furthermore, the cost of large-sized packing columns also increases.

[0086] (3) To optimize the elution buffer usage, under the same extraction conditions and column chromatography method, the elution methods for the solid-phase microextraction column were as follows: elution with 0.5 mL of pure methanol three times, with the eluents combined and diluted to 2 mL for analysis; or elution with 1 mL of pure methanol three times, with the eluents combined and diluted to 5 mL for analysis. The recovery rates of the two elution solvent usage experiments were compared.

[0087] The results of the optimization experiments for different elution methods are shown in Table 8 below.

[0088] Table 8 Under otherwise unchanged conditions, the recovery rate of bile acids was 93.43%-108.49% when eluted three times with 0.5 mL of pure methanol and the eluent was combined and diluted to 2 mL before analysis. The recovery rate was 90.59%-106.39% when eluted three times with 1 mL of pure methanol and the eluent was combined and diluted to 5 mL before analysis. The validation results indicate that excessive methanol eluent is unnecessary for bile acid elution via solid-phase microextraction (SPE). Firstly, it does not improve the recovery rate; secondly, a larger volume of eluent would dilute the concentration of the target metabolites in the sample, requiring more sample volume; and thirdly, large volumes of reagent elution increase experimental costs and pretreatment time.

[0089] Based on the comparison of the above experimental results, the recovery rates of each bile acid substance were calculated. The results showed that the optimal pretreatment step, "solid-phase microextraction column purification," achieved the best recovery rate, with good peak shape, few impurity peaks, and a flat baseline, ranging from 92.99% to 109.79%. Figures 4-11 As shown in the comparison charts, this processing method can enrich some low-content compounds, with good peak shapes, avoiding undetectable or excessively low concentrations due to pretreatment or matrix effects, thus improving detection accuracy. Simultaneously, the solid-phase microextraction column effectively removes small molecule impurities such as salts from the sample, reducing sample contamination of the equipment and column, extending the lifespan of high-value consumables such as the column and ion source needle, and contributing to system stability and the reliability of large-scale sample detection.

[0090] Example 3 This embodiment examines the effect of the solid-phase microextraction column itself on sample extraction and detection.

[0091] 1. Observation experiment.

[0092] Experiment 1: Blank sample (blank unlabeled plasma sample) was extracted according to the extraction steps in Example 1 and then directly loaded onto the instrument without passing through a solid phase microextraction column.

[0093] Experiment 2: Blank sample (blank unlabeled plasma sample) After extraction according to the extraction steps in Example 1, 800 μL of supernatant was extracted by high-speed centrifugation. 3200 mL of pure water was added and mixed well. The mixture was then passed through a Sep-Pak C18 1 cc Vac Cartridg solid-phase microextraction column. The column method was as follows: the activated solid-phase microextraction column was washed with 5 mL of pure water, and the pretreated sample solution obtained earlier was loaded. After rinsing with 1 mL of pure water, the column was eluted three times with 0.5 mL of pure methanol, and the eluents were collected and combined. The volume was then adjusted to 2 mL with pure methanol for instrumental analysis.

[0094] 2. Experimental Results: Analyzing the extracted ion chromatograms of the two sets of experimental data, some examples of substances are given below. Figures 4-11 .

[0095] The comparison results of the control experiment with blank samples show that, compared with the extracted ion chromatograms of some bile acid compounds, column chromatography demonstrates that some compounds are better retained after column chromatography, with flatter baselines and better peak shapes. However, substances such as bile acids, which show little change in response, are also present. Overall, solid-phase microextraction (SPE) columns have advantages for detection and analysis. The significantly lower baseline of the column-coated samples indicates that column chromatography effectively removes impurities introduced during the pretreatment extraction process.

[0096] The optimized pretreatment and purification method obtained from the above embodiments is as follows: The sample is placed in a 2.0 mL centrifuge tube, and 900 μL of extraction buffer (Vacetonitrile:Vmethanol:Vwater = 2:2:1) is added; the centrifuge tube is tightly capped, and the mixture is vortexed for 30 s. The sample tube is then frozen on dry ice for 5 minutes, followed by sonication in an ice-water bath for 5 minutes. The extracted sample is symmetrically placed in a centrifuge and centrifuged at 12000 rpm and 4°C for 15 minutes to remove impurities. The supernatant is then filtered through a 0.22 μm nylon 66 organic phase disposable syringe filter for later use.

[0097] Take 800 μL of the above extract and dilute it with 3200 μL of pure water to prepare the loading solution. Activate the C18 solid-phase microextraction column (Sep-Pak C18 1 cc Vac Cartridge or Sep-Pak C18 3 cc Vac Cartridge) with 5 mL of methanol. Wash the activated solid-phase microextraction column with 5 mL of pure water, load the previously prepared loading solution, rinse with 1 mL of pure water, and then elute three times with 0.5 mL of pure methanol. Collect and combine the eluents, and then make up to 2 mL with pure methanol before instrumental analysis. This method yields the best results, lowers the baseline, and provides more ideal peak shapes and resolutions for bile acids, which is helpful for the qualitative and quantitative analysis of various bile acid compounds.

[0098] Example 4 This embodiment detects bile acid mixed standards under different mobile phases and chromatographic columns, and compares and analyzes the differences in the separation of some difficult-to-separate isomers under different chromatographic conditions to verify the advantages of the preferred chromatographic column and mobile phase formulation of this invention.

[0099] For column comparison, Waters ACQUITY UPLC HSS T3 (100 × 1.0 mm, 1.7 μm) and Waters ACQUITY UPLC BEH C18 (100 × 1.0 mm, 1.7 μm) were selected. Mobile phase selection: Option 1: Phase A: 0.1% formic acid (5mM ammonium acetate), Phase B: pure acetonitrile.

[0100] Option 2: Phase A: 0.01% formic acid, Phase B: pure acetonitrile.

[0101] Option 3: Phase A: 0.1% formic acid (5mM ammonium acetate), Phase B: acetonitrile: methanol = 3:1.

[0102] Elution gradient: T3 column; Phase A: 0.1% formic acid (5 mM ammonium acetate) water; Phase B: B acetonitrile: methanol = 3:1.

[0103] From 0 to 0.5 min, the volume fraction of mobile phase A is 95%, and the remainder is mobile phase B; From 0.5 to 1 min, the volume fraction of mobile phase A changes uniformly from 95% to 75%, with the remainder being mobile phase B; From the first to the fourth minute, the volume fraction of mobile phase A changed uniformly from 75% to 70%, with the remainder being mobile phase B. During the 4th-5th minute, the volume fraction of mobile phase A was 70%, with the remainder being mobile phase B; Between 5 and 6 minutes, the volume fraction of mobile phase A changes uniformly from 70% to 60%, with the remainder being mobile phase B. Between 6 and 8 minutes, the volume fraction of mobile phase A changes uniformly from 60% to 55%, with the remainder being mobile phase B. Between 8 and 11.5 minutes, the volume fraction of mobile phase A changed uniformly from 55% to 30%, with the remainder being mobile phase B. Between 11.5 and 12 minutes, the volume fraction of mobile phase A changed uniformly from 30% to 0%, with the remainder being mobile phase B. During the 12th-13th minute, the volume fraction of mobile phase A was 0%, and the remainder was mobile phase B. From 13 to 13.1 min, the volume fraction of mobile phase A changed from 0% to 95% at a constant rate, with the remainder being mobile phase B; From 13.1 to 15 min, the volume fraction of mobile phase A was 95%, with the remainder being mobile phase B.

[0104] BEH column; Phase A: 0.01% formic acid solution; Phase B: pure acetonitrile.

[0105] From 0 to 0.5 min, the volume fraction of mobile phase A is 90%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90% to 78%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78% to 68%, with the remainder being mobile phase B. From 2.5 to 3.3 min, the volume fraction of mobile phase A was 68%, and the remainder was mobile phase B; Between 3.3 and 5.5 minutes, the volume fraction of mobile phase A changed uniformly from 68% to 64%, with the remainder being mobile phase B. From 5.5 to 9 minutes, the volume fraction of mobile phase A changed uniformly from 64% to 58%, with the remainder being mobile phase B; Between 9 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 58% to 30%, with the remainder being mobile phase B. Between 18 and 19 minutes, the volume fraction of mobile phase A changed uniformly from 30% to 0%, with the remainder being mobile phase B. During the 19th-20th minute, the volume fraction of mobile phase A was 0%, and the remainder was mobile phase B. Between 20 and 20.1 min, the volume fraction of mobile phase A changes uniformly from 0% to 90%, with the remainder being mobile phase B; From 20.1 to 22 min, the volume fraction of mobile phase A was 90%, with the remainder being mobile phase B.

[0106] BEH column, phase A: 0.1% formic acid (5mM ammonium acetate) and water; phase B: pure acetonitrile.

[0107] From 0 to 0.5 min, the volume fraction of mobile phase A is 90%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90% to 78%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78% to 68%, with the remainder being mobile phase B. From 2.5 to 3.3 min, the volume fraction of mobile phase A was 68%, and the remainder was mobile phase B; Between 3.3 and 5.5 minutes, the volume fraction of mobile phase A changed uniformly from 68% to 64%, with the remainder being mobile phase B. From 5.5 to 9 minutes, the volume fraction of mobile phase A changed uniformly from 64% to 58%, with the remainder being mobile phase B; Between 9 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 58% to 30%, with the remainder being mobile phase B. Between 18 and 19 minutes, the volume fraction of mobile phase A changed uniformly from 30% to 0%, with the remainder being mobile phase B. During the 19th-20th minute, the volume fraction of mobile phase A was 0%, and the remainder was mobile phase B. Between 20 and 20.1 min, the volume fraction of mobile phase A changes uniformly from 0% to 90%, with the remainder being mobile phase B; From 20.1 to 22 min, the volume fraction of mobile phase A was 90%, with the remainder being mobile phase B.

[0108] BEH column A phase: 0.1% formic acid (5mM ammonium acetate) water; B phase: acetonitrile: methanol = 3:1.

[0109] From 0 to 0.5 min, the volume fraction of mobile phase A is 90%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90% to 78%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78% to 68%, with the remainder being mobile phase B. From 2.5 to 3.5 min, the volume fraction of mobile phase A was 68%, and the remainder was mobile phase B; Between 3.5 and 5.5 minutes, the volume fraction of mobile phase A changed uniformly from 68% to 64.5%, with the remainder being mobile phase B. Between 5.5 and 9 minutes, the volume fraction of mobile phase A changed uniformly from 64.5% to 58%, with the remainder being mobile phase B. Between 9 and 10 minutes, the volume fraction of mobile phase A changed uniformly from 58% to 57%, with the remainder being mobile phase B. Between 10 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 57% to 33%, with the remainder being mobile phase B. Between 18 and 19.2 minutes, the volume fraction of mobile phase A changed uniformly from 33% to 5%, with the remainder being mobile phase B. From 19.2 to 20.3 min, the volume fraction of mobile phase A was 5%, and the remainder was mobile phase B; Between 20.3 and 20.4 minutes, the volume fraction of mobile phase A changed uniformly from 5% to 90%, with the remainder being mobile phase B. From 20.4 to 23 min, the volume fraction of mobile phase A was 90%, with the remainder being mobile phase B.

[0110] The extracted ion chromatogram of bile acid standard mixture obtained by T3 column extraction is shown (see...). Figure 12 (See Table 9). Several isomers were found to be completely indistinguishable, such as the first four peaks at M / Z=391 and the first two peaks at M / Z=405, resulting in false positives and affecting the accuracy of the qualitative results. Table 9 shows... Figure 12 The peak situation in the middle.

[0111] Table 9 The extracted ion chromatograms of bile acid mixed standards obtained by detection using three mobile phase protocols on a BEH column are shown in (see...). Figure 13 and Table 10, Figure 14 and Table 11, Figure 15 (See Tables 10-12). The preferred embodiment of this invention can effectively separate the first two peaks at M / Z=375, the first four peaks at M / Z=391, and the first two peaks at M / Z=514. Tables 10-12 show... Figures 13-15 The peak situation in the middle.

[0112] Table 10 Table 11 Table 12 At the same time, other bile acid isomers that can be effectively distinguished by the preferred embodiment of the present invention (see...) Figure 16 (As shown in Table 13), these bile acids are relatively easy to elute and separate, with high baseline separation. Table 13 shows... Figure 16 The peak situation in the middle.

[0113] Table 13 Therefore, this invention establishes an efficient extraction and detection method for bile acid samples, enabling the separation and accurate quantification of 50 bile acid compounds, and significantly reducing the false positive problem caused by co-elution of isomer bile acids in traditional detection methods.

[0114] In summary, this invention, through specific column selection, mobile phase formulation optimization, and gradient elution program control, as well as optimization of the pretreatment process, simplifies time-consuming steps such as derivatization and lyophilization while ensuring extraction efficiency and reducing impurity interference. It establishes a highly efficient extraction method for bile acid samples and optimizes chromatographic conditions, thereby achieving the separation and accurate quantification of 50 bile acid compounds (including 40 isomer subtypes). This significantly reduces the false positive problem caused by co-elution of isomer bile acids in traditional detection methods. This method is mainly applied to medical and metabolic research and has extremely high application value.

[0115] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, The method includes: (1) Pre-treat the sample to be tested, then pass it through a C18 solid phase microextraction column, and collect the eluent as the test solution; (2) The test solution and the standard mixture solution were separated and detected by ultra-high performance liquid chromatography-tandem mass spectrometry; the mobile phase used in the ultra-high performance liquid chromatography included: mobile phase A: 0.095-0.105% formic acid aqueous solution containing 4.95-5.05 mM ammonium acetate, mobile phase B: acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol was 3:(0.95-1.05); gradient elution was performed using the mobile phase, and the gradient elution program was as follows: From 0 to 0.5 min, the volume fraction of mobile phase A is 90-90.5%, and the remainder is mobile phase B; Between 0.5 and 1.5 minutes, the volume fraction of mobile phase A changes uniformly from 90-90.5% to 78-78.5%, with the remainder being mobile phase B. Between 1.5 and 2.5 minutes, the volume fraction of mobile phase A changes uniformly from 78-78.5% to 68-68.5%, with the remainder being mobile phase B. During the 2.5-3.5 min interval, the volume fraction of mobile phase A was 68-68.5%, with the remainder being mobile phase B. Between 3.5 and 5.5 minutes, the volume fraction of mobile phase A changes uniformly from 68-68.5% to 64.5-65%, with the remainder being mobile phase B. From 5.5 to 9 minutes, the volume fraction of mobile phase A changed uniformly from 64.5-65% to 58-58.5%, with the remainder being mobile phase B; During the 9th to 10th minute, the volume fraction of mobile phase A changed uniformly from 58-58.5% to 57-57.5%, with the remainder being mobile phase B; Between 10 and 18 minutes, the volume fraction of mobile phase A changed uniformly from 57-57.5% to 33-33.5%, with the remainder being mobile phase B. From 18 to 19.2 min, the volume fraction of mobile phase A changed uniformly from 33-33.5% to 5-5.5%, with the remainder being mobile phase B; From 19.2 to 20.3 min, the volume fraction of mobile phase A was 5-5.5%, with the remainder being mobile phase B; Between 20.3 and 20.4 minutes, the volume fraction of mobile phase A changed uniformly from 5-5.5% to 90-90.5%, with the remainder being mobile phase B. From 20.4 to 23 min, the volume fraction of mobile phase A was 90-90.5%, with the remainder being mobile phase B; (3) Linear regression equations for each bile acid subtype compound were established by mixing standard samples of different concentrations for quantitative calculation.

2. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (1), the sample pretreatment steps include: The collected sample was mixed with the extract, frozen with dry ice, and then shaken to promote sample lysis. The sample was extracted by ultrasonic extraction in an ice-water bath, frozen with dry ice, and then centrifuged to collect the supernatant. The supernatant was filtered, and the filtrate was collected. The filtrate was mixed with water and passed through a C18 solid-phase microextraction column. After rinsing with pure water, the sample was eluted with pure methanol at least three times. The eluent was collected as the test solution.

3. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that, The extract is an acetonitrile-methanol-water solution, wherein the volume ratio of acetonitrile, methanol and water is (1.9-2.1):(1.9-2.1):(0.9-1.1); Preferably, the dry ice freezing time is 4-6 minutes; Preferably, the oscillation conditions are: oscillation at 1200-1250 rpm for 10-15 minutes; Preferably, the ultrasonic extraction step takes 8-12 minutes; Preferably, the centrifugation step is performed at a speed of 11,000-12,000 rpm and a temperature of 0-4℃ for 8-12 minutes. Preferably, the filtration uses a disposable needle filter with a 0.22 μm nylon 66 membrane.

4. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 2 or 3, characterized in that, The volume ratio of the filtrate to water is 1:(3.5-4.5); Preferably, the packing material of the C18 solid-phase microextraction column is Sep-Pak C18 1 cc Vac Cartridge.

5. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1-4, characterized in that, In step (2), the ultra-high performance liquid chromatography column is a Waters ACQUITY UPLCBEH C18 column, with a size of 1.7 μm and a diameter of 2.1 mm × 100 mm; the column temperature is 34-36℃.

6. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1-5, characterized in that, In step (2), the ultra-high performance liquid chromatograph is an ACQUITY UPLC I-Class PLUS, Waters, with an injection volume of 1.8-2.2 μL.

7. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1-6, characterized in that, In step (2), the mass spectrometry conditions are as follows: an electrospray ionization (ESI) source is used, and negative ion mode is used for detection. The instrument parameters are as follows: curtain gas is 35 psi, ion spray voltage is -4500V, ion source temperature is 550℃, nebulizing gas is 50 psi, and auxiliary gas is 55 psi.

8. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1-7, characterized in that, In step (2), the standard mixed solution includes: dehydrolithocholic acid, isolithocholic acid, lithocholic acid, 2,3-demethyldeoxycholic acid, 7-ketolithocholic acid, 12-ketolithocholic acid, procholic acid, ursodeoxycholic acid, porcine deoxycholic acid, chenodeoxycholic acid, deoxycholic acid, isodeoxycholic acid, dehydrocholic acid, 7,12-diketolithocholic acid, 7-ketodeoxycholic acid, 12-dehydrocholic acid, 3-dehydrocholic acid, ursolic acid, α-mouse cholic acid, β-mouse cholic acid, porcine cholic acid, allocholic acid, cholic acid, glycinelithocholic acid, glycineursodeoxycholic acid, and glycine. Aminodeoxycholic acid, glycine deoxycholic acid, glycine dehydrocholic acid, glycine cholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, iso-allolithocholic acid, mouse deoxycholic acid, isursodeoxycholic acid, iso-sugar deoxycholic acid, 3β-deoxycholic acid, norcholic acid, 3β-cholic acid, ω-mouse cholic acid, glycine chenodeoxycholic acid, lithocholic acid sulfate, glycine cholic acid, taurolectocholic acid, taurolectocholic acid, taurolectocholic acid, and chenodeoxycholic acid-3-β-glucuronic acid.

9. The high-throughput detection method for bile acids based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 8, characterized in that, The concentration range of each bile acid subtype standard in the standard mixed solution is 0.1-5000 ng / mL.

10. The application of the high-throughput bile acid detection method based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1-9 in the detection of bile acids.

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