Detection method of bile acid metabolism marker group and kit thereof

Abstract

The invention provides a bile acid metabolism marker group detection method and a kit thereof. The method comprises the following steps: collecting a sample, preparing a reagent, preparing a sample to be detected, and carrying out liquid chromatography-tandem mass spectrometry detection and analysis. The preparation of the sample to be detected comprises the steps of protein precipitation, centrifugation and dilution. According to the method, 55 types of multi-source bile acids can be simply, conveniently and quickly detected in a high-throughput and wide-coverage manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and medical devices, and particularly relates to a detection method of a bile acid metabolism marker group and a kit thereof. BACKGROUND

[0002] Bile acids (BAs) are a class of important lipid molecules derived from cholesterol, whose biosynthesis mainly occurs in the liver through multiple enzymatic reactions. The human bile acid pool has significant chemical diversity, mainly including: primary bile acids: synthesized directly by hepatocytes, including cholic acid (CA) and chenodeoxycholic acid (CDCA); secondary bile acids: metabolically transformed by intestinal flora, such as deoxycholic acid (DCA) and lithocholic acid (LCA).

[0003] From the molecular structure, bile acids can be divided into: free bile acids, conjugated bile acids (combined with glycine, taurine, sulfate, glucuronic acid, etc.) Among them, conjugated bile acids are formed under the catalysis of bile acid-coenzyme A-amino acid N-acyltransferase (BAAT), which has better water solubility and biological activity. It is worth noting that there is a unique murine cholic acid (MCA) metabolic pathway in rodents.

[0004] As an important signaling molecule, bile acids participate in the regulation of body metabolism through multiple pathways: nuclear receptor pathway: mainly through the activation of FXR, PXR, etc. Nuclear receptors regulate lipid and glucose metabolism; membrane receptor pathway: through TGR5 receptor to participate in the regulation of energy homeostasis and inflammatory response; microbe-host interaction: intestinal flora modifies bile acids through deconjugation, dehydroxylation, etc. Reaction to form a complex metabolic network.

[0005] Bile acid metabolism disorder is closely related to various diseases: metabolic diseases: obesity, diabetes, non-alcoholic fatty liver; hepatobiliary diseases: cholestasis, cholelithiasis; inflammatory diseases: intestinal inflammation, systemic inflammatory response. Therefore, establishing a precise bile acid detection method is of great significance for understanding the pathogenesis of these diseases and developing new diagnosis and treatment strategies.

[0006] In the diagnosis and monitoring of clinical hepatobiliary and intestinal diseases, traditional enzymatic analysis method usually only measures the total bile acid (Total Bile Acids, TBA) level as a reference index. However, bile acids (Bile Acids, BAs) are a class of structurally diverse endogenous metabolites, and the level changes of different subtypes are closely related to specific diseases (such as cholestasis, cirrhosis, intestinal liver circulation disorder, etc.). With the development of precision medicine, relying only on total bile acid detection cannot meet the needs of disease typing, mechanism research and individualized treatment. By precisely monitoring the concentration changes of different bile acid subtypes, disease subtypes can be more effectively distinguished, disease progression can be assessed, and the basis for targeted treatment can be provided.

[0007] Current common BAs detection techniques include: enzymatic method (total bile acid determination), gas chromatography (GC), radioimmunoassay (RIA), high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS) and the like.

[0008] Although LC-MS / MS has become the current mainstream method due to its high sensitivity and high specificity, it still faces the following challenges in practical application:

[0009] (1) Difficulty in separation due to structural diversity: Different bile acid subtypes have significant differences in physicochemical properties due to different number of hydroxyl groups, positions and binding modes (free type vs. glycine / taurine conjugated type vs. sulfated type). The pKa span is large (1.5~5.6): the sulfate group (pKa~1.5) needs to be stable at low pH, while the free carboxyl group (pKa~5.0) is easy to ionize at high pH. It partially ionizes (COO - / COOH coexists) at pH 4.0, resulting in double peaks or peak shape distortion, ultimately leading to decreased quantitative accuracy, especially for low concentration free bile acids (such as UDCA), which have a significant impact on peak shape tailing / leading. The LogP range is wide (-2.2~+3.0): strong hydrophilic sulfated type (TCA-S, LogP=-2.2) and strong hydrophobic lithocholic acid (LCA, LogP=3.0) require completely opposite chromatographic conditions, and the retention time window is insufficient. A single analysis method is difficult to optimize the separation and detection of all subtypes, resulting in low detection throughput.

[0010] (2) Isomer interference: multiple subtypes have structurally similar isomers (such as isomers UDCA / CDCA, GCA / GCDCA), which have similar LogP (ΔLogP<0.5) and small ΔpKa (ΔpKa<0.3). Conventional chromatography is difficult to effectively separate. The risk of false qualitative is high, which affects the discrimination of disease-related markers (such as CDCA elevation indicating bile stasis).

[0011] (3) Taurine type and sulfated type bile acids are sensitive to pH: taurine conjugated type and sulfate conjugated type bile acids are highly sensitive to the pH of the elution system. The sulfate group remains stable at low pH (such as <3.5), but is easily hydrolyzed at high pH (such as >7.0). While the taurine conjugated type and other subtypes (as well as free bile acids) are prone to peak tailing at low pH. This significant peak difference (sulfate hydrolysis vs. other subtypes tailing) under different pH conditions results in poor compatibility of the analysis method for different bile acid subtypes. The optimal pH requirements of the sulfate group (pKa~1.5) and other subtypes conflict. Ultimately, the recovery rate of sulfated type (such as TCA-S) is low, and the risk of missed detection is high.

[0012] (4) Sample differences affect the universality of the method: The bile acid profiles of different species are significantly different. For example, mice are mainly bound to MCA and bezoar, accounting for 30%, and require pH ≤ 3.0. Human samples are mainly bound to CA and glycine, and can be relaxed to pH 3.5. Therefore, cross-species studies need to re-validate the method and optimize the method accordingly.

[0013] The existing technology has the following limitations:

[0014] Low throughput and insufficient specificity: Most methods can only detect a limited number of bile acids (usually <15), making it difficult to cover a wide range of bile acid subtypes and to exclude interference from isomers.

[0015] Limited sample applicability: Existing technologies are mostly designed for specific species and specific samples (such as human serum, feces / intestinal contents), and are difficult to extend to different species and different sample types, such as complex samples like tissues and cells.

[0016] Complex preprocessing: Some methods rely on cumbersome sample preprocessing (such as derivatization and lyophilization), which affects detection efficiency.

[0017] CN111830161A discloses a method for detecting 15 bile acids in serum. This method is based on UPLC-MS / MS technology, which directly precipitates serum proteins with methanol (without derivatization). Using an ACQUITY UPLC BEH C18 column (35-45℃) with a mobile phase of 0.01-0.1% formic acid aqueous solution / methanol (flow rate 0.3-0.5 mL / min), the 15 bile acids are separated ultra-rapidly in 6.5 minutes. Quantification is performed using an isotope internal standard method, making it suitable for high-throughput serum screening in clinical practice. The main limitations include: precision needs improvement (CV% is close to the 15% threshold), insufficient validation of isomer separation and detection and interference effects, limited detection range (only 15), and inability to extend to other biological samples or species, thus limiting its clinical application.

[0018] CN111257476A discloses a method for detecting 17 bile acids in serum using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). This method, based on HPLC-MS / MS technology, uses a methanol-acetonitrile mixture to precipitate serum proteins, followed by a 0.01% formic acid-5mM ammonium acetate / methanol gradient elution system (total time 10 minutes) to achieve efficient separation and quantification of the 17 bile acids. Its advantages include simple and rapid operation, strong resistance to matrix interference (isotope internal standard correction), and low cost, making it suitable for routine clinical testing. However, it has significant limitations: the number of detectable species is limited (only 17), and the isomer separation capability and clinical applicability have not been fully validated, making it difficult to extend to the analysis of multi-species or complex samples.

[0019] CN118409034A discloses a method for detecting bile acid subtypes, which is based on HPLC-QTOF to detect 15 bile acid subtypes (including free and glycine / taurine bound subtypes). Advantages: High-resolution mass spectrometry (accurate to 4 decimal places), suitable for solution samples. Limitations: Lacks the specificity of MRM mode and cannot be applied to complex biological samples.

[0020] CN114894926A discloses a bile acid detection method based on the dried blood speckle method, which uses the dried blood speckle method combined with LC-MS / MS to detect bile acids (≤10 types) in human blood samples. Advantages: High sample stability, suitable for remote collection. Limitations: Cumbersome pretreatment steps (ultrasonic incubation, freeze centrifugation), and cannot be extended to tissue or cross-species samples.

[0021] CN106885867A discloses a method for detecting five taurine-bound bile acids in serum using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Limitations: It only covers a limited number of subtypes, and the lyophilization step significantly reduces throughput.

[0022] CN111474256A discloses a method for quantitative analysis of bile acids in fecal / intestinal contents based on HPLC-MS / MS. This method quantifies 28 bile acids in fecal / intestinal contents using HPLC-MS / MS. Advantages: Covers major human bile acid subtypes. Limitations: Does not cover other species or low-abundance samples (such as blood, tissue, and cells).

[0023] Gómez C et al. provided an LC-MS / MS method for detecting 36 bile acids in serum and liver tissue (Gómez C, Stücheli S, Kratschmar DV, Bouitbir J, Odermatt A. Development and Validation of a Highly Sensitive LC-MS / MS Method for the Analysis of Bile Acids in Serum, Plasma, and Liver Tissue Samples. Metabolites. 2020 Jul 9;10(7):282). Its advantages include coverage of major subtypes in humans and rodents. Its limitations include complex and time-consuming sample pretreatment, including dilution, extraction, incubation, centrifugation, double extraction, drying, and reconstitution, with each sample processing session taking 2-4 hours.

[0024] Currently, LC-MS / MS remains the most promising technology platform, enabling the separate determination of multiple bile acid isomers and allowing for bile acid loading tests and the determination of sulfate-bound bile acids in urine. However, further optimization of chromatographic separation, mass spectrometry scanning modes, and sample pretreatment strategies is needed to achieve a comprehensive understanding of the bile acid metabolic network. Developing a method and reagents based on HPLC-MS / MS technology in multiple reaction monitoring mode for the simultaneous detection of >50 bile acid isomers, including isomers, with high sensitivity and specificity, a wide detection range, compatibility with blood, urine, fecal / intestinal contents, cell, and tissue samples from multiple species, and simple pretreatment procedures to ensure detection efficiency, is a pressing issue in this field. Summary of the Invention

[0025] One object of the present invention is to provide a method for detecting a group of bile acid metabolism markers.

[0026] Another object of the present invention is to provide a kit for detecting bile acid metabolism biomarkers.

[0027] This invention first provides a method for detecting bile acid metabolism biomarkers, comprising the following steps: sample collection, reagent preparation, sample preparation, liquid chromatography-tandem mass spectrometry detection and analysis;

[0028] The steps for preparing the test sample include: protein precipitation, centrifugation, and dilution;

[0029] The chromatographic method for detection by liquid chromatography-tandem mass spectrometry employs gradient elution of mobile phase A and mobile phase B; mobile phase A is a 1-5 mM aqueous solution of ammonium acetate with a pH of 5-7; mobile phase B is methanol (preferably, mobile phase A is a 2 mM aqueous solution of ammonium acetate with a pH of 6.8; mobile phase B is methanol).

[0030] The gradient elution flow rate is 0.2-0.4 mL / min (preferably, the gradient elution flow rate is 0.3 mL / min).

[0031] The bile acid metabolism markers include: lithocholic acid, chenodeoxycholic acid, porphyrin deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, taurol chenodeoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol β-mouse cholic acid, taurol porphyrin deoxycholic acid, porphyrin deoxycholic acid, glycolithocholic acid, glycolithocholic acid, 6-ketolithocholic acid, 7-ketolithocholic acid, 12-ketoursodeoxycholic acid, 7-ketodeoxycholic acid, 3-dehydrocholic acid, chenodeoxycholic acid, deoxylithocholic acid, ω-mouse cholic acid, λ-mouse cholic acid, and allocholic acid. Taurol ω-mouse cholic acid, taurol α-mouse cholic acid, taurol λ-mouse cholic acid, glycol λ-mouse cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycololcholic acid sulfate-bound, glycololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, and taurolcholic acid sulfate-bound; one or more combinations thereof;

[0032] The chromatographic column used in the chromatography is a water-resistant reverse-phase C18 column;

[0033] The mass spectrometry for the liquid chromatography-tandem mass spectrometry detection uses an electrospray ionization source, a negative ion mode for detection, and a multiple reaction detection (MRP) scan mode.

[0034] The method described is not intended for the direct diagnosis or treatment of disease.

[0035] According to a specific embodiment of the present invention, preferably, in the above detection method, the bile acid metabolism markers include: lithocholic acid, chenodeoxycholic acid, porcine deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolol ...

[0036] In the above detection method, preferably, the water-resistant reverse-phase C18 chromatographic column is selected from Venusil MP C18 column with a column length of 50-150 mm, an inner diameter of 3-5 mm, and a packing material of 1.7-5 μm, or a chromatographic column with equivalent performance (more preferably selected from Venusil MP C18 column with a column length of 100 mm, an inner diameter of 4.6 mm, and a packing material of 2.6 μm, or a chromatographic column with equivalent performance).

[0037] In the above detection method, preferably, the gradient elution conditions are as follows:

[0038] From 0 to 1 minute, the volume percentage of mobile phase A was maintained at 95%, and the volume percentage of mobile phase B was maintained at 5%.

[0039] Over 1-1.2 minutes, the volume percentage of mobile phase A decreased from 95% to 55%, while the volume percentage of mobile phase B increased from 5% to 45%.

[0040] 1.2-2 min, the volume percentage of mobile phase A is maintained at 55%, and the volume percentage of mobile phase B is maintained at 45%;

[0041] Within 2-4 minutes, the volume percentage of mobile phase A decreased from 55% to 38%, while the volume percentage of mobile phase B increased from 45% to 62%.

[0042] For 4-9 minutes, the volume percentage of mobile phase A remained at 38%, and the volume percentage of mobile phase B remained at 62%.

[0043] Within 9-12 minutes, the volume percentage of mobile phase A decreased from 38% to 30%, while the volume percentage of mobile phase B increased from 62% to 70%.

[0044] For 12-14.5 min, the volume percentage of mobile phase A was maintained at 30%, and the volume percentage of mobile phase B was maintained at 70%.

[0045] Over 14.5-16 minutes, the volume percentage of mobile phase A decreased from 30% to 20%, while the volume percentage of mobile phase B increased from 70% to 80%.

[0046] For 16-19 minutes, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%.

[0047] Over 19-20 minutes, the volume percentage of mobile phase A decreased from 20% to 10%, while the volume percentage of mobile phase B increased from 80% to 90%.

[0048] For 20-22 minutes, the volume percentage of mobile phase A was maintained at 10%, and the volume percentage of mobile phase B was maintained at 90%.

[0049] Over 22-24 minutes, the volume percentage of mobile phase A decreased from 10% to 2%, while the volume percentage of mobile phase B increased from 90% to 98%.

[0050] For 24-26 minutes, the volume percentage of mobile phase A was maintained at 2%, and the volume percentage of mobile phase B was maintained at 98%.

[0051] Over 26-27 minutes, the volume percentage of mobile phase A increased from 2% to 40%, while the volume percentage of mobile phase B decreased from 98% to 60%.

[0052] Over 27-28 minutes, the volume percentage of mobile phase A increased from 40% to 95%, while the volume percentage of mobile phase B decreased from 60% to 5%.

[0053] For 28-30 minutes, the volume percentage of mobile phase A was maintained at 95%, and the volume percentage of mobile phase B was maintained at 5%.

[0054] The chromatographic column temperature was 30℃, the injector temperature was 10℃, the injection volume was 5μL, and the needle washing solution was 50% methanol aqueous solution.

[0055] In the above detection method, preferably, the multi-reaction detection scanning strategy is segmented scanning; the temperature of the electrospray ion source is 450~550℃, the voltage is -4000~-4500V, the collision gas is set to medium, the atomizing gas flow rate is 45~65, the desolventizing gas flow rate is 45~65, and the backflushing gas flow rate is 30~40 (more preferably, the temperature of the electrospray ion source is 550℃, the voltage is -4500V, the collision gas is set to medium, the atomizing gas flow rate is 55, the desolventizing gas flow rate is 55, and the backflushing gas flow rate is 30).

[0056] In the above detection method, preferably, the sample to be tested is selected from one or more combinations of serum, plasma, urine, bile, feces, intestinal contents, cells, and tissues; for example, the sample to be tested is tissue, and the tissue is homogenized before protein precipitation. The tissue homogenization includes: adding a homogenizing agent to the tissue, grinding and homogenizing to obtain a homogenized sample to be tested; the homogenizing agent is a pre-cooled 50% methanol aqueous solution.

[0057] In the above detection method, preferably, the protein precipitation includes: adding a precipitant and an internal standard to the sample to be tested, vortexing for 5-10 minutes (more preferably 10 minutes) to obtain protein precipitation; the precipitant is a mixed solution of acetonitrile and isopropanol with 1%-5% ammonia added, wherein the volume ratio of acetonitrile to isopropanol is (5-8):(2-5); the internal standard is a 30-60% acetonitrile solution of deuterated bile acid (more preferably, the internal standard is a 50% acetonitrile solution of deuterated bile acid); the sample to be tested is cells, which are vortexed for 30-60 seconds and ultrasonically disrupted before vortexing;

[0058] The deuterated bile acid derivatives include: lithocholic acid, chenodeoxycholic acid, porphyrin deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, taurol chenodeoxycholic acid, taurol porphyrin deoxycholic acid, taurol ursodeoxycholic acid, taurol deoxycholic acid, taurol β-mouse cholic acid, taurol cholic acid, porphyrin deoxycholic acid, glycolithocholic acid, glycolithocholic acid, 6-ketolithocholic acid, 7-ketolithocholic acid, 12-ketoursodeoxycholic acid, 7-ketodeoxycholic acid, 3-dehydrocholic acid, chenodeoxycholic acid, deoxylithocholic acid, ω-mouse cholic acid, λ-mouse cholic acid, allocholic acid, taurol cholic acid. A deuterated product of one or more of the following: ω-mouse cholic acid, taurine α-mouse cholic acid, taurine λ-mouse cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycololcholic acid sulfate-bound, glycochenodeoxycholic acid sulfate-bound, glycoursodeoxycholic acid sulfate-bound, glycololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurine chenodeoxycholic acid sulfate-bound, taurine ursodeoxycholic acid sulfate-bound, taurine deoxycholic acid sulfate-bound, and tauronic acid sulfate-bound.

[0059] In the above detection method, preferably, the centrifugation and dilution include: precipitating the protein at 4-10℃, centrifuging at 2000-4000g for 10-15 min, separating the supernatant, transferring 100-200 μL of the supernatant to a clean 96-well V-plate, adding an equal volume of 5% (v / v) methanol aqueous solution containing 2-5 mM ammonium acetate for dilution, and vortexing for 30-60 s to obtain the diluted test sample (more preferably, precipitating the protein at 4℃, centrifuging at 4000g for 10 min, separating the supernatant, transferring 100-200 μL of the supernatant to a clean 96-well V-plate, adding an equal volume of 5% (v / v) methanol aqueous solution containing 2 mM ammonium acetate for dilution, and vortexing for 30 s to obtain the diluted test sample).

[0060] In the above detection method, preferably, the preparation of reagents includes: restoring the calibrator, quality control sample, and internal standard to room temperature; reconstituteing the calibrator and quality control sample in purified water to the concentration required for the calibration curve; vortexing for 30-60 s before use; pre-cooling the precipitant; and sonicating mobile phase A and mobile phase B for 1-5 min each (more preferably, vortexing for 30 s before use; pre-cooling the precipitant; and sonicating mobile phase A and mobile phase B for 1 min each).

[0061] The standard and quality control products include: lithocholic acid, chenodeoxycholic acid, porcine deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolol ... A powder containing one or more of the following: taurine, taurine α-cholic acid, taurine λ-cholic acid, glycolamine λ-cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycolamine lithocholic acid sulfate-bound, glycolamine chenodeoxycholic acid sulfate-bound, glycolamine ursodeoxycholic acid sulfate-bound, glycolamine cholic acid sulfate-bound, glycolamine cholic acid sulfate-bound, tauramine lithocholic acid sulfate-bound, tauramine chenodeoxycholic acid sulfate-bound, tauramine ursodeoxycholic acid sulfate-bound, tauramine cholic acid sulfate-bound, and tauramine cholic acid sulfate-bound.

[0062] In the above detection method, preferably, the analysis includes:

[0063] Calibration curve plotting: Add the internal standard to the calibrator and standard at the required concentrations for the calibration curve, and detect them using liquid chromatography-tandem mass spectrometry. Obtain the peak areas of the chromatograms and mass spectra of the calibrator and standard, and the corresponding peak areas of the internal standard. Use the concentration of the calibrator as the independent variable x. i The mean ratio of the peak areas of the corresponding concentration calibrators and internal standards is used as the dependent variable y. i Calculate the linear regression equation y = ax + b and the correlation coefficient r;

[0064] Quality control data analysis: After the calibration curve r≥0.990, the signal intensity of the quality control sample is substituted into the regression equation to obtain the concentration of the quality control sample;

[0065] Sample data analysis: After the test results of the quality control sample are within the expected range, the signal intensity obtained from the liquid chromatography-tandem mass spectrometry detection of the sample is substituted into the regression equation to obtain the concentration of the target substance in the sample.

[0066] The present invention also provides a kit for detecting bile acid metabolism biomarkers, the kit containing calibrators, quality control materials, internal standards, precipitants, homogenizers, mobile phase A and mobile phase B and diluents as described in the above detection method, the kit having a detection limit of 0.25 ng / mL and a quantitation limit of 0.5 ng / mL.

[0067] This invention offers the following beneficial technical effects: It provides a simple, rapid, high-throughput, and broad-coverage method and kit for detecting bile acid metabolism biomarkers. This method is based on a high-aqueous-phase reversed-phase chromatography system using HPLC-MS / MS technology, employing negative ion scanning combined with multiple reaction monitoring (MRM) mode to simultaneously separate and detect 55 bile acid subtypes (including isomers and polar metabolites), encompassing the separation and detection of multiple isomers, thus avoiding quantitative biases caused by insufficient separation in traditional methods. Simultaneously, it exhibits high sensitivity and specificity, and a wide detection range, for the first time covering the detection of bile acids in serum, plasma, bile, liver / intestinal tissues, and intestinal contents, with cross-species applicability to humans, mice, and cattle. Furthermore, it overcomes the problems of low throughput, limited sample applicability, complex sample pretreatment, and unsuitability for clinical and research translational applications in existing detection technologies, providing a truly one-stop cross-sample analysis platform for clinical and research purposes. Attached Figure Description

[0068] Figure 1 The LC-MS / MS spectra of taurine cholic acid (trihydroxy) and taurine deoxycholic acid (dihydroxy) isomers under gradient elution conditions in Example 1 are shown.

[0069] Figure 2 The LC-MS / MS spectra of deoxycholic acid and glycine-type deoxycholic acid isomers under gradient elution conditions in Example 1 are shown.

[0070] Figure 3 The LC-MS / MS spectra of deoxycholic acid and glycine-type deoxycholic acid isomers under gradient elution conditions in Comparative Example 1 are shown.

[0071] Figure 4 The LC-MS / MS spectra of a series of isomers of taurine cholic acid (trihydroxy) and taurine deoxycholic acid (dihydroxy) under gradient elution conditions in Comparative Example 1 are shown.

[0072] Figure 5 LC-MS / MS spectra of bile acids eluted with 2 mM ammonium formate aqueous solution.

[0073] Figure 6LC-MS / MS spectra of bile acids eluted with 5 mM ammonium formate aqueous solution.

[0074] Figure 7 LC-MS / MS spectra of bile acids eluted with 2 mM ammonium formate + 0.1% formic acid aqueous solution.

[0075] Figure 8 LC-MS / MS spectra of bile acids eluted with 5 mM ammonium acetate aqueous solution.

[0076] Figure 9 The LC-MS / MS chromatogram is obtained by elution with 2 mM ammonium acetate aqueous solution (pH 6.8).

[0077] Figure 10 The LC-MS / MS chromatogram is obtained by elution with 5 mM ammonium acetate aqueous solution (pH 6.8).

[0078] Figure 11 The LC-MS / MS spectrum is obtained by elution with 7.5 mM ammonium acetate aqueous solution (pH 6.8).

[0079] Figure 12 The LC-MS / MS chromatogram is obtained by elution of aqueous phase with 5 mM ammonium acetate + 0.01% formic acid aqueous solution (pH 5.6).

[0080] Figure 13 The LC-MS / MS chromatogram is obtained by elution with an aqueous phase of 5 mM ammonium acetate + ammonia solution (pH 7.8).

[0081] Figure 14 This refers to the pretreatment process for mammalian blood and tissue samples.

[0082] Figure 15 This is an LC-MS / MS spectrum of serum bile acids (some subtypes) extracted using acetonitrile.

[0083] Figure 16 The image shows the LC-MS / MS spectra of serum bile acids (partial subtypes) extracted based on acetonitrile:isopropanol (1:1, v / v).

[0084] Figure 17 This is an LC-MS / MS chromatogram of fetal bovine serum bile acids (some subtypes) extracted based on acetonitrile:isopropanol.

[0085] Figure 18 This is an LC-MS / MS chromatogram of fetal bovine serum bile acids (some subtypes) extracted based on acetonitrile:isopropanol (1% ammonia).

[0086] Figure 19 The image shows the LC-MS / MS spectra of fetal bovine serum bile acids (some subtypes) extracted using acetonitrile:isopropanol (2% ammonia).

[0087] Figure 20 The image shows the LC-MS / MS spectra of fetal bovine serum bile acids (some subtypes) extracted using acetonitrile:isopropanol (5% ammonia).

[0088] Figure 21 The extracted ion chromatograms of 25 bile acids in mouse serum are shown.

[0089] Figure 22 MRM detection profiles of dihydroxy and monohydroxy bile acid subtypes (including isomers) in mouse serum samples.

[0090] Figure 23 The extracted ion chromatograms of 25 bile acids from mouse liver tissue are shown.

[0091] Figure 24 MRM detection profiles of dihydroxy and monohydroxy bile acid subtypes (including isomers) in mouse liver tissue samples.

[0092] Figure 25 This is a chromatogram of total ion currents of bile acids in mouse intestinal tissue.

[0093] Figure 26 MRM detection profiles of dihydroxy and monohydroxy bile acid subtypes (including isomers) in mouse intestinal tissue samples.

[0094] Figure 27A A series of glycine-bound trihydroxycholic acid profiles in human plasma.

[0095] Figure 27B This is a series of chromatograms of glycine-bound trihydroxycholic acid in mouse plasma.

[0096] Figure 28A A series of taurine-bound dihydroxycholic acid profiles in human plasma.

[0097] Figure 28B This is a series of taurine-bound dihydroxycholic acid profiles in mouse plasma. Detailed Implementation

[0098] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0099] It should be noted that, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0100] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0101] It should be understood that the terms “comprising,” “including,” and / or “containing” as used herein specify the presence of the stated features, integers, steps, components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0102] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0103] (a) Optimization of LC-MS / MS conditions

[0104] 1. Chromatographic gradient optimization

[0105] This invention utilizes a reversed-phase C18 column (Venusil MP C18, Waters BEH C18, or other equivalent columns) with high aqueous phase tolerance (100% water compatibility) to achieve the joint determination of bile acids in different LogP ranges and baseline separation of a series of isomers through fractional gradient elution. The differences in baseline separation performance under different conditions are compared in Example 1 and Comparative Example 1.

[0106] The gradient elution conditions for Example 1 are shown in Table 1.

[0107] Table 1

[0108]

[0109] The gradient elution conditions for Comparative Example 1 are shown in Table 2.

[0110] Table 2

[0111]

[0112] Example 1: LC-MS / MS chromatograms of taurine cholic acid (trihydroxy) and taurine deoxycholic acid (dihydroxy) isomers under gradient elution conditions are shown below. Figure 1 As shown. The LC-MS / MS spectra of deoxycholic acid and glycosaminoglycine isomers under gradient elution conditions in Example 1 are shown below. Figure 2 As shown. The LC-MS / MS spectra of deoxycholic acid and glycine-type deoxycholic acid isomers under gradient elution conditions in Comparative Example 1 are shown below.Figure 3 As shown. The LC-MS / MS chromatograms of a series of isomers of taurine cholic acid (trihydroxy) and taurine deoxycholic acid (dihydroxy) under gradient elution conditions in Comparative Example 1 are shown below. Figure 4 As shown in the results, in Example 1, the peak shape and resolution of the free deoxycholic acid isomers were good, and the resolution of the glycine-bound deoxycholic acid isomers was also good. The peak shapes of the ox gall-bound cholic acid (trihydroxy series) and ox gall-bound deoxycholic acid (dihydroxy series) isomers were good, and baseline separation could be achieved. Compared to Example 1, under the conditions of Comparative Example 1, the resolution and peak shape of the different epimers of TCA were poor. Baseline separation could not be achieved between DCA and CDCA. Figure 3 UDCA and its isomers cannot be separated and detected due to interference. Figure 4 ).

[0113] 2. Mobile phase optimization

[0114] This invention systematically investigated the effects of different buffer systems on the separation efficiency and mass spectrometry response of target bile acids. The ammonium formate and ammonium acetate buffer systems were compared, including: 2 mM ammonium formate aqueous solution, 5 mM ammonium formate aqueous solution, 2 mM ammonium formate + 0.1% formic acid aqueous solution, and 5 mM ammonium acetate aqueous solution. The LC-MS / MS spectra of bile acids eluted with 2 mM ammonium formate aqueous solution are shown below. Figure 5 As shown; the LC-MS / MS spectrum of bile acids eluted with 5 mM ammonium formate aqueous solution is as follows. Figure 6 As shown; the LC-MS / MS chromatogram of bile acids eluted with 2 mM ammonium formate + 0.1% formic acid aqueous solution is as follows. Figure 7 As shown; the LC-MS / MS spectrum of bile acids eluted with 5 mM ammonium acetate aqueous solution is as follows. Figure 8 As shown.

[0115] The results showed that in the ammonium formate system, taurine-bound bile acid molecules had poor peak shape and co-eluted with interferences, making effective separation impossible. Using the ammonium acetate system significantly improved the chromatographic resolution and mass spectrometry response of the target bile acids, resulting in good overall performance.

[0116] Further detailed investigations were conducted on ammonium acetate buffer systems at different concentrations and pH values: 2 mM, 5 mM, and 7.5 mM ammonium acetate aqueous solutions (all pH 6.8), 5 mM ammonium acetate + 0.01% formic acid aqueous solution (pH 5.6), and 5 mM ammonium acetate + ammonia aqueous solution (pH 7.8). The LC-MS / MS chromatograms of the aqueous phase eluted with 2 mM ammonium acetate aqueous solution (pH 6.8) are shown below. Figure 9 As shown; the LC-MS / MS spectrum of the aqueous phase eluted with 5 mM ammonium acetate aqueous solution (pH 6.8) is as follows. Figure 10As shown; the LC-MS / MS spectrum of the aqueous phase eluted with 7.5 mM ammonium acetate aqueous solution (pH 6.8) is as follows. Figure 11 As shown; the LC-MS / MS chromatogram of aqueous phase eluted with 5 mM ammonium acetate + 0.01% formic acid aqueous solution (pH 5.6) is as follows. Figure 12 As shown; the LC-MS / MS chromatogram of the aqueous phase eluted with 5 mM ammonium acetate + ammonia solution (pH 7.8) is as follows. Figure 13 As shown.

[0117] The results showed that when the mobile phase was 2 mM ammonium acetate aqueous solution (pH 6.8), the target bile acids showed the best performance in chromatographic resolution (key isomer separation factor >1.5) and mass spectrometry response intensity (average signal enhancement ≥30%). In particular, the response of sulfate-bound bile acids was enhanced by about 40% compared with acidic conditions, so it was determined to be the optimal aqueous mobile phase condition.

[0118] 3. Optimization of mass spectrometry detection conditions

[0119] This invention employs electrospray ionization (ESI) and multiple reaction monitoring (MRM) scanning in negative ion mode for mass spectrometry detection. To optimize the performance of simultaneous multi-component analysis, a novel schedule-based segmented scanning strategy is applied. This strategy dynamically allocates the mass spectrometry dwell time to its corresponding peak window based on the precise chromatographic retention time of each target analyte. This significantly increases the effective number of scans and ion acquisition efficiency of each detection channel, thereby greatly enhancing the signal response intensity (sensitivity) of the target compound. Simultaneously, by focusing the acquisition of target ion pairs within a specific time window, background noise and interference from co-eluting components in non-target time periods are effectively reduced, significantly improving the detection's anti-interference capability and method selectivity. The ion source parameters are shown in Table 3.

[0120] Table 3

[0121]

[0122] MRM ion pairs and ionization parameters are shown in Tables 4-6 (Tables 5-6 are continuations of Table 4).

[0123] Table 4

[0124]

[0125] Table 5

[0126]

[0127] Table 6

[0128]

[0129] (II) Construction and optimization of LC-MS / MS pretreatment system for bile acid detection in biological samples

[0130] For complex biological samples such as blood, tissues, and cells, this invention systematically optimizes the pretreatment method. By precisely screening the components of the lysis buffer, the formulation and ratio of the protein precipitant, and adjusting the pH of the extraction system, the extraction efficiency of the target analyte is improved. The pretreatment process for mammalian blood and tissue samples is as follows: Figure 14 As shown.

[0131] 2. Optimization of the extractant system in protein precipitants

[0132] To maximize the extraction efficiency of bile acids from biological samples, this invention systematically screened a variety of solvent systems, covering:

[0133] Methanol-based: Methanol, Methanol:Isopropanol (3:7, v / v), Methanol:Isopropanol:Water (2:7:1 and 7:2:1, v / v / v)

[0134] Acetonitrile group: acetonitrile, acetonitrile:isopropanol (1:1 and 3:7, v / v), acetonitrile:isopropanol:water (4:5:1, v / v / v)

[0135] The LC-MS / MS signal intensities of serum bile acids extracted with different extractants are shown in Table 7.

[0136] Table 7

[0137]

[0138] Serum bile acids (some subtypes) extracted with acetonitrile LC-MS / MS chromatograms are shown below. Figure 15 As shown. The LC-MS / MS chromatograms of serum bile acids (partial subtypes) extracted using acetonitrile:isopropanol (1:1, v / v) are shown below. Figure 16 As shown in the figure. Based on a comprehensive comparison of the response values ​​of bile acids in the serum matrix by LC-MS / MS, acetonitrile:isopropanol (1:1, v / v) was finally determined as the optimal extraction solvent formulation. This formulation significantly improved the extraction recovery rate of the target analyte, reflected in an average increase of approximately 40% in the detection sensitivity of LC-MS / MS.

[0139] 2. System optimization of acid-base regulators in protein precipitants

[0140] Based on the preferred extractant acetonitrile:isopropanol (1:1, v / v), the effects of adding different acid-base regulators on the extraction efficiency of bile acids were systematically investigated. The acid-base regulators evaluated included:

[0141] Acidity regulators: 0.01% (v / v) formic acid, 0.1% (v / v) formic acid

[0142] Salt / buffer: 5 mM ammonium acetate

[0143] Alkalinity regulator: ammonia (1%, 2%, 5% (v / v))

[0144] The effects of adding different acid-base regulators on the LC-MS / MS signal intensity of bile acids extracted by the preferred extractant acetonitrile:isopropanol (1:1, v / v) are shown in Table 8.

[0145] Table 8

[0146]

[0147] LC-MS / MS chromatograms of fetal bovine serum bile acids (partial subtypes) extracted with acetonitrile:isopropanol are shown below. Figure 17 As shown; LC-MS / MS chromatograms of fetal bovine serum bile acids (partial subtypes) extracted based on acetonitrile:isopropanol (1% ammonia) are shown below. Figure 18 As shown; LC-MS / MS chromatograms of fetal bovine serum bile acids (partial subtypes) extracted based on acetonitrile:isopropanol (2% ammonia) are shown below. Figure 19 As shown; LC-MS / MS chromatograms of fetal bovine serum bile acids (partial subtypes) extracted based on acetonitrile:isopropanol (5% ammonia) are shown below. Figure 20 As shown in the results, based on a systematic comparison of the overall response values ​​of bile acids in the samples using LC-MS / MS, 5% (v / v) ammonia was determined to be the optimal acid-base regulator. This optimization significantly improved the extraction efficiency and detection sensitivity of the target bile acids.

[0148] Ultimately, the optimized protein precipitant was a solution with a volume ratio of acetonitrile:isopropanol:5% ammonia of 19:19:2.

[0149] 3. Optimization of diluent

[0150] This invention investigated the effects of dilution operations and diluents on detection performance. Four methods were used to dilute the supernatant after centrifugation of the precipitate, including: water, water (containing 2 mM ammonium acetate), 5% (v / v) methanol / water solution (containing 2 mM ammonium acetate), and 50% (v / v) methanol / water solution (containing 2 mM ammonium acetate).

[0151] Based on LC-MS / MS observations, dilution with 5% (v / v) methanol / water solution (containing 2 mM ammonium acetate) significantly improved the ion suppression effect and enhanced the signal intensity of the target analyte, with an average signal enhancement of approximately 40%.

[0152] The effects of different dilution solutions on the signal intensity of extracted bile acids by LC-MS / MS are shown in Table 9.

[0153] Table 9

[0154]

[0155] This invention simplifies the process by optimizing the pretreatment method, significantly improving the extraction efficiency of the target analyte. Blood samples require only a single extraction step for direct injection, with sample consumption as low as 20 μL. For the prepared sample, the optimized complex solution system effectively eliminates potential solvent effects, significantly improving chromatographic peak shape. Simultaneously, this strategy significantly reduces co-extraction of matrix interferences, helping to mitigate matrix effects and greatly enhancing the compatibility and stability of the detection system. Tissue sample processing is equally efficient, requiring only 10 mg of sample. After grinding and homogenization, and a single-step protein precipitation, dilution is sufficient for injection, resulting in a simple and efficient process.

[0156] (III) Design and testing of the reagent kit

[0157] 1. Reagent kit design

[0158] The optimized reagent formulation and reaction system conditions were used to develop the detection kit. The kit contains calibrators, quality control materials, internal standards, homogenizers, precipitants, and mobile phase additives, and can be used to detect 100 samples. The main components are shown in Table 10.

[0159] Table 10

[0160]

[0161] Among them, 25 bile acids are lithocholic acid (LCA), chenodeoxycholic acid (UDCA), porphyrin deoxycholic acid (HDCA), ursodeoxycholic acid (CDCA), deoxycholic acid (DCA), α-mouse cholic acid (αMCA), β-mouse cholic acid (βMCA), cholic acid (CA), glycolithocholic acid (GLCA), glycolithocholic acid (GUDCA), glycolithocholic acid (GCDCA), glycolithocholic acid (GDCA), glycolithocholic acid (GCA), and taurol lithocholic acid (T). Taurine chenodeoxycholic acid (TUDCA), taurine sucrase deoxycholic acid (THDCA), taurine ursodeoxycholic acid (TCDCA), taurine deoxycholic acid (TDCA), tauronic acid (TCA), glycosaminoglycan deoxycholic acid (GHDCA), 6-ketolithocholic acid (6-KLCA), 7-ketolithocholic acid (7-KLCA), taurine ω-mouse cholic acid (TωMCA), 12-ketodeoxycholic acid (12-KDCA), and demethyldeoxycholic acid (NDCA).

[0162] 2. Test applications of the reagent kit

[0163] This kit is used for the in vitro detection of the levels of various bile acid metabolites in mammalian serum, plasma, tissue and cell samples, and features high sensitivity, high specificity and wide dynamic range.

[0164] Bile acids are important physiological mediators in the human body's internal environment. They mainly include cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA), as well as their corresponding glycine- and bezoar-conjugated forms, totaling 15 types. Abnormal bile acid metabolism in the human body is associated with the occurrence of gallstones, atherosclerosis, hypertension, and many other diseases. The detection of serum bile acid composition and levels is of significant clinical research value for understanding the metabolic characteristics of bile acids in the body.

[0165] Bile acids are a collective term for cholanonic acids present in bile, encompassing various structurally similar steroid compounds. They can be classified into primary and secondary bile acids based on their origin. Primary bile acids (including cholic acid and chenodeoxycholic acid) are synthesized in liver cells using cholesterol as a raw material. Secondary bile acids (including deoxycholic acid, lithocholic acid, and trace amounts of ursodeoxycholic acid) are then formed in the intestines through the action of enzymes in intestinal bacteria. Bile acids are primarily secreted into bile from the liver in conjugated form. Conjugated bile acids refer to those formed by the combination of the aforementioned bile acids with glycine or taurine. Abundant conjugated bile acids include glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid, as well as smaller amounts of glycolithocholic acid, glycoursodeoxycholic acid, taurolithocholic acid, and tauroursodeoxycholic acid.

[0166] Whether free or bound, bile acids contain both hydrophilic (hydroxyl, carboxyl, sulfonyl) and hydrophobic (methyl and hydrocarbon) groups within their molecules, exhibiting strong interfacial activity. This activity promotes the formation of mixed micro-macromolecules of lipids, which plays a crucial role in the digestion and absorption of lipids and maintaining cholesterol solubility in bile. Therefore, bile acids have important physiological functions in the body, including emulsifying fats, producing bile, and preventing gallstone formation. Current research has found that various hepatobiliary diseases can cause enterohepatic circulation disorders of bile acids, leading to abnormal serum bile acid levels. This abnormality also affects the liver's synthesis and secretion of bile acids. Therefore, serum bile acids are an important marker for assessing the normality of hepatobiliary function. Currently, in the clinical diagnosis and monitoring of hepatobiliary and enteric diseases, the level of total bile acids (including free and bound bile acids, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid) is often used as a reference indicator. With the rapid development of precision medicine, monitoring total bile acid levels is insufficient for the accurate diagnosis of related diseases and the effective correction of anticipated diseases. Measuring the levels of multiple bile acid subtypes in the body is of great significance for understanding the overall metabolic level and status of bile acids, as well as for research on related physiological and pathological mechanisms.

[0167] 3. Testing principle of the reagent kit

[0168] This kit uses high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) for highly sensitive and specific quantitative or relative quantitative detection of target analytes. The principle is as follows:

[0169] First, liquid chromatography (LC) separation is performed: After sample pretreatment, separation is achieved using a reversed-phase C18 column. Gradient elution of the mobile phase (water / organic phase) allows analytes of different polarities to elute sequentially according to their retention time differences, effectively eliminating matrix interference. The chromatographic eluents are then ionized using an electrospray ionization (ESI) source, forming charged ions (such as [M+H+)) under a high-voltage electric field. + Or [MH] - The sample is fed into a mass spectrometry system. In tandem mass spectrometry (MS / MS), the first-stage mass spectrometer (Q1) screens for the precursor ion of the target ion. The precursor ion enters the collision cell and undergoes collision-induced dissociation (CID), fragmenting to generate the characteristic product ion. The second-stage mass spectrometer (Q3) specifically monitors the product ion, achieving highly selective quantification through multiple reaction monitoring (MRM) mode, significantly reducing background noise. Based on the peak area ratio of the target ion to the internal standard, combined with single-point or multi-point calibration methods, the concentration of the analyte in the sample is calculated.

[0170] 4. Sample requirements for the reagent kit

[0171] This kit is suitable for mammalian serum, plasma, tissue, and cell samples. Serum and plasma samples should be at least 100 μL, urine samples at least 1 mL, tissue samples at least 20 mg, and cell samples at least 1 × 10⁻⁶. 6 .

[0172] Sample collection and preparation:

[0173] (1) Sample type

[0174] Bile acid detection is applicable to the following biological samples: serum / plasma (heparin sodium or EDTA anticoagulation is recommended, avoid using heparin lithium), urine (morning urine or 24-hour urine is recommended), feces / intestinal contents (must be homogenized before sampling), bile (obtained surgically or by puncture, oxidation must be avoided), and cells / tissues (preserved after flash freezing in liquid nitrogen).

[0175] (2) Collection requirements

[0176] Serum / Plasma: Blood should be collected on an empty stomach (8-12 hours of fasting) to avoid hemolysis. Heparin sodium or EDTA is recommended for anticoagulation; avoid using lithium heparin. Centrifuge within 30 minutes of blood collection (4°C, 3000×g, 10 minutes) to separate serum and plasma.

[0177] Urine: It is recommended to collect morning urine or 24-hour urine. Centrifuge within 30 minutes after collection (4℃, 2000×g, 5 minutes) to remove precipitate and collect the supernatant. It is recommended to add 0.1% NaN3 (sodium azide) as a preservative.

[0178] Feces / intestinal contents: After homogenization, samples are taken and stored at -80°C immediately after collection, or a stabilizer (such as RNAlater) is added, mixed, and then aliquoted.

[0179] Bile / tissue: Obtained surgically or by puncture, oxidation must be avoided, and the sample should be flash-frozen in liquid nitrogen after collection to avoid repeated freeze-thaw cycles.

[0180] Cells: After digestion, centrifuge the cells, wash them with PBS at least twice, count them, centrifuge the lower layer of cells, and flash freeze them in liquid nitrogen to avoid repeated freeze-thaw cycles.

[0181] (3) Storage and transportation

[0182] Short-term storage: Stable for ≤48 hours at 2-8℃, and stable for 6 months when frozen at -80℃. Aliquot into small volumes (e.g., 100 μL / tube) to reduce freeze-thaw cycles.

[0183] Transportation conditions: Dry ice transportation (ensure temperature remains below -60℃ throughout the journey) to avoid temperature fluctuations.

[0184] (4) Precautions

[0185] Avoid repeated freeze-thaw cycles (≤2 times is recommended), as this may lead to bile acid degradation. Do not use blood collection tubes containing antioxidants (such as BHT), as this may interfere with testing. Fecal / intestinal contents samples must be labeled with the collection time and storage conditions.

[0186] Note: The above standards are based on routine bile acid metabolomics studies; special projects require separate optimization. Specific stability may vary depending on the type of bile acid; verification before testing is recommended.

[0187] 5. Detection methods of the kit

[0188] (1) Reagent preparation before testing:

[0189] Calibrators and quality control samples: Remove the calibrators and quality control samples from the frozen kit, bring them to room temperature, add the reconstituted volume of purified water, and vortex for 30 seconds before use.

[0190] Internal standards: Remove the internal standards from the frozen kit, bring them to room temperature, and vortex for 30 seconds before use.

[0191] Precipitant: Remove the precipitant from the cryopreservation kit and pre-cool before use.

[0192] Mobile phase: Take out mobile phase A and add it to purified water at a ratio of 1:1000. Sonicate for 1 min to prepare mobile phase A.

[0193] Take out mobile phase B, sonicate for 1 min, and prepare mobile phase B.

[0194] (2) Sample processing and testing procedures for serum, plasma, urine, bile samples, calibrators, and quality control materials:

[0195] 1) Add sample: Accurately pipette 50-100 μL of calibrator / quality control sample / sample, add 5 μL of standard solution and 100-200 μL of precipitant, vortex for 10 min to obtain the precipitated sample.

[0196] 2) Centrifugation: Centrifuge the precipitated sample at 4℃ and 4000g for 10 min, separate the supernatant, and obtain the supernatant liquid;

[0197] 3) Detection: Transfer 100-200 μL of supernatant to a clean 96-well V-plate, add an equal volume of diluent to dilute, vortex mix for 30 s, and then perform LC-MS / MS detection.

[0198] (3) Sample processing and testing procedures:

[0199] 1) Tissue homogenization: Accurately weigh a certain amount (20-100 mg) of tissue sample, add 200 μL of pre-cooled homogenizing solution, grind and homogenize at 60 Hz for 80 s to obtain the homogenized sample.

[0200] 2) Add sample: Take 100 μL of homogenized sample, add 5 μL of standard solution and 400 μL of precipitant, vortex for 10 min to obtain precipitated sample.

[0201] 3) Centrifugation: Centrifuge the precipitated sample at 4 ℃ and 4000 g for 10 min, separate the supernatant, and obtain the supernatant liquid;

[0202] 4) Detection: Transfer 100-200 μL of supernatant to a clean 96-well V-plate, add an equal volume of diluent to dilute, vortex mix for 30 s, and then perform LC-MS / MS detection.

[0203] (4) Cell sample processing and detection procedures:

[0204] 1) Cell disruption: To ≥10 6 Add 5 μL of internal standard solution and 200 μL of pre-cooled precipitant to the cell pellet, vortex for 30 s, sonicate to disrupt, and vortex for 10 min to obtain the precipitated sample.

[0205] 2) Centrifugation: Centrifuge the precipitated sample at 4 ℃ and 4000 g for 10 min, separate the supernatant, and obtain the supernatant liquid;

[0206] 3) Detection: Transfer 100-200 μL of supernatant to a clean 96-well V-plate, add an equal volume of diluent to dilute, vortex mix for 30 s, and then perform LC-MS / MS detection.

[0207] (5) Data analysis and results:

[0208] 1) Calibration curve plotting: using the concentration of the calibrator as the independent variable x i The mean ratio of the peak areas of the corresponding concentration calibrator and internal standard is used as the dependent variable y. i Calculate the linear regression equation y = ax + b and the correlation coefficient r;

[0209] 2) Quality control sample data analysis: When the r of the calibration curve is ≥ 0.990, the signal intensity of the quality control sample is substituted into the regression equation to obtain the concentration of the quality control sample;

[0210] 3) Sample data analysis: When the test results of the quality control product are within the expected range, the test signal of the sample is substituted into the regression equation to obtain the concentration of the target substance in the sample.

[0211] (iv) Performance evaluation of the reagent kit

[0212] The performance evaluation of the detection method provided by this invention mainly includes detection specificity, sensitivity, and precision to ensure that the expected results are achieved. The requirements for test samples, solvent preparation, and sample preparation procedures in the method evaluation were all completed according to the operating instructions provided in the kit in section (III).

[0213] 1. Evaluation of linearity of standard curve

[0214] Experimental procedure:

[0215] Calibration samples were prepared according to the blank matrix spiking method. The analyte concentration was plotted on the x-axis, and the peak area ratio of the analyte to the internal standard was plotted on the y-axis. A weighted average (W=1 / X) was then calculated. 2 Linear regression is performed using the least squares method to obtain the linear regression equation (Y=bX+a). The correlation coefficient r is calculated, and r ≥ 0.99 is required.

[0216] Experimental results and conclusions:

[0217] The linear results for 25 bile acids are shown in Table 11.

[0218] Table 11

[0219]

[0220] According to the linear range set by the method debugging, the correlation coefficient R of the standard curve is ≥0.990, and the evaluation of the standard curve meets the methodological requirements.

[0221] 2. Evaluation of Limit of Detection (LOD) and Limit of Quantification (LOQ)

[0222] Experimental procedure:

[0223] Low-concentration spiked samples (close to the lowest concentration of the standard curve) were prepared using a blank matrix, with three replicates per concentration. LC-MS / MS analysis was performed, and the signal-to-noise ratio (S / N) was recorded. LOD: Concentrations corresponding to S / N ≥ 3; LOQ: Concentrations with S / N ≥ 10 and meeting the accuracy (80-120%) and precision (RSD ≤ 20%) requirements. The LOQ concentration was validated six times.

[0224] Note: LOQ should be less than or equal to the lowest concentration of the standard curve. B% is the percentage of deviation.

[0225] Experimental results and conclusions:

[0226] The limits of detection and limits of quantitation for 25 bile acids are shown in Table 12.

[0227] Table 12

[0228]

[0229] Experimental data show that the method can achieve a detection limit of 0.25 ng / mL and a quantitation limit of 0.5 ng / mL for each bile acid subtype, which can meet the needs of biological sample detection.

[0230] 3. Precision and accuracy evaluation

[0231] Experimental procedure:

[0232] Two quality control samples, one low and one high, were prepared in parallel for each concentration, with five samples prepared. Each sample was tested once to evaluate the intra-lot precision (RSD) and accuracy (RE). Three batches were tested consecutively to evaluate the inter-batch precision (cv%).

[0233] Experimental results and conclusions:

[0234] The precision and accuracy results for 25 bile acids are shown in Tables 13-20 (Tables 14-20 are continuations of Table 13).

[0235] Table 13

[0236]

[0237] Table 14

[0238]

[0239] Table 15

[0240]

[0241] Table 16

[0242]

[0243] Table 17

[0244]

[0245] Table 18

[0246]

[0247] Table 19

[0248]

[0249] Table 20

[0250]

[0251] Precision and accuracy test data from three consecutive batches showed that the intra-batch / inter-batch precision and accuracy of this method for detecting each bile acid subtype were ≤15%, meeting the requirements for biological sample detection.

[0252] 4. Test results of reagent kit samples

[0253] Mouse serum and liver tissue samples were tested using a kit. Figure 21 The figure shows the extracted ion chromatograms of 25 bile acids in mouse serum. As can be seen from the figure, the peaks are symmetrical and sharp, indicating good separation. The 25 target bile acids are evenly distributed within the set detection time period, demonstrating that the method has excellent separation efficiency and analytical stability.

[0254] Figure 22 This study presents the MRM (Matching Resolved Biomarkers) profiles of dihydroxy and monohydroxy bile acid subtypes (including isomers) in mouse serum samples. The results show that even structurally similar isomers can be baseline separated under these detection conditions, demonstrating the method's high resolution for complex bile acid subtypes.

[0255] Figure 23 This is an extracted ion chromatogram of 25 bile acids from mouse liver tissue. The peak shapes and retention time distributions of each component in the chromatogram are good, indicating that the liver tissue sample pretreatment and chromatographic conditions are suitable for the simultaneous detection of multiple bile acids.

[0256] Figure 24 This image shows the MRM (Morphological Resonance Mapping) profiles of dihydroxy and monohydroxy bile acid isoforms in mouse liver tissue samples (including isomers). The image demonstrates baseline separation between all isomers, further validating the good selectivity and specificity of this method in the liver tissue matrix. Figure 25 This is the total ion chromatogram of bile acids in mouse intestinal tissue. The chromatogram shows that, within the complex matrix of intestinal tissue, the peaks of each bile acid component remain sharp and symmetrical, and their distribution is reasonable throughout the analysis time. This indicates that the detection method can effectively handle the complexity of intestinal samples, achieving efficient separation and detection of multiple bile acids.

[0257] Figure 26 This image shows the MRM (Matching Resonance Mapping) spectra of dihydroxy and monohydroxy bile acid subtypes (including isomers) in mouse intestinal tissue samples. As can be seen from the image, even structurally similar isomers achieved clear baseline separation under these detection conditions, demonstrating the accuracy and reliability of this method for the qualitative and quantitative identification of bile acid subtypes in intestinal tissue samples.

[0258] The fingerprint profiles of glycine-bound and taurine-bound bile acids were compared and analyzed using a kit to detect human and mouse plasma samples.

[0259] Figure 27A A series of glycine-bound trihydroxycholic acid profiles in human plasma. Figure 27BThis is a chromatogram of glycine-bound trihydroxycholic acids in mouse plasma. Comparative results show that the chromatographic peak responses of this series of bile acids are significant in human plasma, with a greater variety and abundance.

[0260] Figure 28A A series of taurine-bound dihydroxycholic acid profiles in human plasma. Figure 28B This is a chromatogram of taurine-bound dihydroxycholic acids in mouse plasma. The contrasting results show that mouse plasma exhibits a significant advantage in this series of bile acids, with both the number and intensity of peaks being significantly higher than in human plasma.

[0261] In summary, the spectral analysis clearly reveals significant differences in bile acid metabolism patterns among different species: glycine-conjugated bile acids are predominant in human plasma, while taurine-conjugated bile acids are absolutely dominant in mouse plasma. This finding provides important experimental evidence for cross-species bile acid metabolism research.

[0262] The above embodiments illustrate and describe the main features and advantages of the present invention in detail. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A method for detecting a group of bile acid metabolism markers, characterized in that, Includes the following steps: Sample collection, reagent preparation, sample preparation for testing, liquid chromatography-tandem mass spectrometry detection and analysis; The steps for preparing the test sample include: protein precipitation, centrifugation, and dilution; The chromatographic method for detection by liquid chromatography-tandem mass spectrometry employs gradient elution with mobile phase A and mobile phase B; mobile phase A is a 1-5 mM ammonium acetate aqueous solution with a pH of 5-7; mobile phase B is methanol. The gradient elution flow rate is 0.2-0.4 mL / min; The bile acid metabolism markers include: lithocholic acid, chenodeoxycholic acid, porphyrin deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, taurol chenodeoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol porphyrin deoxycholic acid, taurol β-mouse cholic acid, taurol porphyrin deoxycholic acid, porphyrin deoxycholic acid, glycolithocholic acid, glycolithocholic acid, 6-ketolithocholic acid, 7-ketolithocholic acid, 12-ketoursodeoxycholic acid, 7-ketodeoxycholic acid, 3-dehydrocholic acid, chenodeoxycholic acid, deoxylithocholic acid, ω-mouse cholic acid, λ-mouse cholic acid, and allocholic acid. Taurol ω-mouse cholic acid, taurol α-mouse cholic acid, taurol λ-mouse cholic acid, glycol λ-mouse cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycololcholic acid sulfate-bound, glycololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, glycolololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, and taurolcholic acid sulfate-bound; one or more combinations thereof; The chromatographic column used in the chromatography is a water-resistant reverse-phase C18 column; The mass spectrometry for the liquid chromatography-tandem mass spectrometry detection uses an electrospray ionization source, a negative ion mode for detection, and a multiple reaction detection (MRP) scan mode. The method described is not intended for the direct diagnosis or treatment of disease.

2. The detection method according to claim 1, characterized in that, The water-resistant reverse-phase C18 chromatographic column is selected from Venusil MP C18 columns with a column length of 50-150 mm, an inner diameter of 3-5 mm, and a packing material of 1.7-5 μm, or a chromatographic column with equivalent performance.

3. The detection method according to claim 1, characterized in that, The gradient elution conditions are as follows: From 0 to 1 minute, the volume percentage of mobile phase A was maintained at 95%, and the volume percentage of mobile phase B was maintained at 5%. Over 1-1.2 minutes, the volume percentage of mobile phase A decreased from 95% to 55%, while the volume percentage of mobile phase B increased from 5% to 45%. 1.2-2 min, the volume percentage of mobile phase A is maintained at 55%, and the volume percentage of mobile phase B is maintained at 45%; Within 2-4 minutes, the volume percentage of mobile phase A decreased from 55% to 38%, while the volume percentage of mobile phase B increased from 45% to 62%. For 4-9 minutes, the volume percentage of mobile phase A remained at 38%, and the volume percentage of mobile phase B remained at 62%. Within 9-12 minutes, the volume percentage of mobile phase A decreased from 38% to 30%, while the volume percentage of mobile phase B increased from 62% to 70%. For 12-14.5 min, the volume percentage of mobile phase A was maintained at 30%, and the volume percentage of mobile phase B was maintained at 70%. Over 14.5-16 minutes, the volume percentage of mobile phase A decreased from 30% to 20%, while the volume percentage of mobile phase B increased from 70% to 80%. For 16-19 minutes, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%. Over 19-20 minutes, the volume percentage of mobile phase A decreased from 20% to 10%, while the volume percentage of mobile phase B increased from 80% to 90%. For 20-22 minutes, the volume percentage of mobile phase A was maintained at 10%, and the volume percentage of mobile phase B was maintained at 90%. Over 22-24 minutes, the volume percentage of mobile phase A decreased from 10% to 2%, while the volume percentage of mobile phase B increased from 90% to 98%. For 24-26 minutes, the volume percentage of mobile phase A was maintained at 2%, and the volume percentage of mobile phase B was maintained at 98%. Over 26-27 minutes, the volume percentage of mobile phase A increased from 2% to 40%, while the volume percentage of mobile phase B decreased from 98% to 60%. Over 27-28 minutes, the volume percentage of mobile phase A increased from 40% to 95%, while the volume percentage of mobile phase B decreased from 60% to 5%. For 28-30 minutes, the volume percentage of mobile phase A was maintained at 95%, and the volume percentage of mobile phase B was maintained at 5%. The chromatographic column temperature was 30℃, the injector temperature was 10℃, the injection volume was 5μL, and the needle washing solution was 50% methanol aqueous solution.

4. The detection method according to claim 1, characterized in that, The multi-reaction detection scanning strategy is segmented scanning; the temperature of the electrospray ion source is 450~550℃, the voltage is -4000~-4500V, the collision gas is set to medium, the atomizing gas flow rate is 45~65, the desolventizing gas flow rate is 45~65, and the backflushing gas flow rate is 30~40.

5. The detection method according to claim 1, characterized in that, The sample to be tested is selected from one or more combinations of serum, plasma, urine, bile, feces, intestinal contents, cells, and tissues.

6. The detection method according to claim 1, characterized in that, The protein precipitation process includes: adding a precipitant and an internal standard to the sample to be tested, vortexing for 5-10 minutes to obtain protein precipitation; the precipitant is a mixed solution of acetonitrile and isopropanol with 1%-5% ammonia added, wherein the volume ratio of acetonitrile to isopropanol is (5-8):(2-5); the internal standard is a 30-60% acetonitrile solution of bile acid deuterated product; the sample to be tested is cells, which are vortexed for 30-60 seconds and ultrasonically disrupted before vortexing; The deuterated bile acid derivatives include: lithocholic acid, chenodeoxycholic acid, porphyrin deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, glycolithocholic acid, taurol chenodeoxycholic acid, taurol porphyrin deoxycholic acid, taurol ursodeoxycholic acid, taurol deoxycholic acid, taurol β-mouse cholic acid, taurol cholic acid, porphyrin deoxycholic acid, glycolithocholic acid, glycolithocholic acid, 6-ketolithocholic acid, 7-ketolithocholic acid, 12-ketoursodeoxycholic acid, 7-ketodeoxycholic acid, 3-dehydrocholic acid, chenodeoxycholic acid, deoxylithocholic acid, ω-mouse cholic acid, λ-mouse cholic acid, allocholic acid, taurol cholic acid. A deuterated product of one or more of the following: ω-mouse cholic acid, taurine α-mouse cholic acid, taurine λ-mouse cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycololcholic acid sulfate-bound, glycochenodeoxycholic acid sulfate-bound, glycoursodeoxycholic acid sulfate-bound, glycololcholic acid sulfate-bound, taurololcholic acid sulfate-bound, taurine chenodeoxycholic acid sulfate-bound, taurine ursodeoxycholic acid sulfate-bound, taurine deoxycholic acid sulfate-bound, and tauronic acid sulfate-bound.

7. The detection method according to claim 1, characterized in that, The centrifugation and dilution process includes: precipitating the protein at 4-10℃, centrifuging at 2000-4000g for 10-15 min, separating the supernatant, transferring 100-200 μL of the supernatant to a clean 96-well V-plate, adding an equal volume of 5% (v / v) methanol aqueous solution containing 2-5 mM ammonium acetate for dilution, and vortexing for 30-60 s to obtain the diluted sample to be tested.

8. The detection method according to claim 1, characterized in that, The preparation of the reagents includes: restoring the calibrators, quality control samples, and internal standards to room temperature; reconstituted the calibrators and quality control samples in purified water to the concentration required for the calibration curve; vortexing for 30-60 seconds before use; pre-cooling the precipitant; and sonicating mobile phase A and mobile phase B for 1-5 minutes each. The standard and quality control products include: lithocholic acid, chenodeoxycholic acid, porcine deoxycholic acid, ursodeoxycholic acid, deoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, cholic acid, glycolol ... A powder containing one or more of the following: taurine, taurine α-cholic acid, taurine λ-cholic acid, glycolamine λ-cholic acid, 12-ketodeoxycholic acid, demethyldeoxycholic acid, sulfate-bound lithocholic acid, sulfate-bound chenodeoxycholic acid, sulfate-bound ursodeoxycholic acid, sulfate-bound deoxycholic acid, sulfate-bound cholic acid, glycolamine lithocholic acid sulfate-bound, glycolamine chenodeoxycholic acid sulfate-bound, glycolamine ursodeoxycholic acid sulfate-bound, glycolamine cholic acid sulfate-bound, glycolamine lithocholic acid sulfate-bound, tauramine chenodeoxycholic acid sulfate-bound, tauramine ursodeoxycholic acid sulfate-bound, tauramine cholic acid sulfate-bound, and tauramine cholic acid sulfate-bound.

9. The detection method according to claim 1, characterized in that, The analysis includes: Calibration curve plotting: Add the internal standard to the calibrator and standard at the required concentrations for the calibration curve, and detect them using liquid chromatography-tandem mass spectrometry. Obtain the peak areas of the chromatograms and mass spectra of the calibrator and standard, and the corresponding peak areas of the internal standard. Use the concentration of the calibrator as the independent variable x. i The mean ratio of the peak areas of the corresponding concentration calibrators and internal standards is used as the dependent variable y. i Calculate the linear regression equation y = ax + b and the correlation coefficient r; Quality control data analysis: After the calibration curve r≥0.990, the signal intensity of the quality control sample is substituted into the regression equation to obtain the concentration of the quality control sample; Sample data analysis: After the test results of the quality control sample are within the expected range, the signal intensity obtained from the liquid chromatography-tandem mass spectrometry detection of the sample is substituted into the regression equation to obtain the concentration of the target substance in the sample.

10. A kit for detecting bile acid metabolism markers, characterized in that, The kit contains calibrators, quality control samples, internal standards, precipitants, homogenizers, mobile phase A and mobile phase B, and diluents as described in any one of claims 1-9. The limit of detection of the kit is 0.25 ng / mL, and the limit of quantitation is 0.5 ng / mL.

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