LC-MS / MS method for detecting bile acid in biological sample

Abstract

The invention discloses an LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method for detecting bile acid in a biological sample, which adopts a liquid chromatography-tandem mass spectrometry method and comprises the following steps: S1, constructing a database, separating isomeride, negatively optimizing bile or excrement overload substances, S2, optimizing biological sample extraction conditions, S3, making a standard curve, and S4, performing chromatography-mass spectrometry detection and analysis. The biological sample is a blood sample, a bile sample, a tissue sample or an excrement sample. According to the invention, 60 bile acids are detected by adopting an LC-MS / MS method, and complete biological sample types are covered. According to the method, mass spectrum parameter negative optimization is carried out on 16 bile acids which often reach the upper limit of mass spectrum response in bile and excrement, meanwhile, high and low concentrations of bile acids are quantified, and the traditional thinking that the sample content is too high and exceeds the upper limit of mass spectrum and needs to be diluted once or multiple times is broken through. According to the method, pretreatment is simple, and rapid extraction of various bile acids is achieved on the premise that the sensitivity of the method is not affected.

Description

Technical Field

[0001] The present invention relates to the technical field of bile acid detection, and particularly relates to an LC-MS / MS method for detecting bile acids in biological samples. Background Art

[0002] Bile acids are a class of complex substances with a cholestane nucleus and are essential substances for human fat metabolism. Primary bile acids are synthesized in the liver, and secondary bile acids are produced under the action of intestinal microorganisms. Abnormal changes in the content of different types of bile acids are usually related to diseases such as the liver and intestines. Therefore, accurately detecting the content of bile acids is of great significance for the diagnosis and treatment of diseases. Currently, the main detection methods for bile acids are liquid chromatography, capillary electrophoresis, gas chromatography (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. It has experienced the development from total content detection to qualitative and quantitative analysis of single species of single substances. Due to the complex and diverse structures of bile acids, the early liquid chromatography had low sensitivity and inaccurate qualitative analysis, and was gradually replaced by gas chromatography (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, the GC-MS method requires sample derivatization to make it easy to vaporize for detection, and the pretreatment is relatively complex. While LC-MS / MS does not require derivatization and has relatively high sensitivity and precision, and is the most commonly used detection method in research.

[0003] The problems faced in current bile acid detection mainly include the following three aspects. First, there are a large number of bile acid types with complex structures, which pose great challenges to their separation and detection. Second, current detection methods are mostly for the detection of single or several bile acids in a single sample type, lacking detection methods for multiple sample types and multiple bile acids. Different bile acids have different diagnostic significance in clinical practice. For example, the metabolic changes of CA (cholic acid) and DCA (deoxycholic acid) are related to the progression of gastric cancer. Therefore, detecting the levels of each bile acid in the body rather than simply quantitatively measuring the total bile acid level is of great significance for disease screening, diagnosis, and differentiation. Third, the content of some bile acids in fecal and bile samples is relatively high, and conventional detection often reaches the upper limit of instrument detection, requiring sample dilution and multiple tests to accurately quantitatively detect. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention uses the LC-MS / MS method to quantitatively detect 60 bile acids in common biological samples such as blood, liver tissue, bile, and feces. The mobile phase components and composition of this method have been improved, enabling baseline separation of 49 bile acid isomers in 14 groups. It has the advantages of simple pretreatment (without complex pretreatment steps such as derivatization and solid-phase extraction), a large number of detected bile acids, comprehensive coverage of biological sample types, good separation performance, short analysis time, etc. Moreover, for 16 bile acids with high content in bile and feces and often reaching the upper limit of mass spectrometry response, negative optimization of mass spectrometry parameters has been carried out. Both the original quantitative ion pairs and the negatively optimized quantitative ion pairs are retained in the detection method, enabling simultaneous detection and quantitative determination of high-concentration and low-concentration bile acids in one test, breaking the traditional thinking that when the sample content is too high and exceeds the upper limit of mass spectrometry, it needs to be diluted once or multiple times and requires multiple runs on the machine for detection, and has the advantages of convenient operation and more accurate and reliable quantitative results.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: An LC-MS / MS method for detecting bile acids in biological samples, which uses liquid chromatography-tandem mass spectrometry for detection, and includes the following steps: S1 construction of a database, separation of isomers, and negative optimization of overloaded substances in bile or feces; S2 optimization of the extraction conditions of biological samples; S3 preparation of a standard curve; S4 chromatographic and mass spectrometric detection and analysis. The biological samples are blood samples, bile samples, tissue samples, or fecal samples. Step S1 includes: S11: Take bile acid standards, prepare a stock solution of the standards, and use the method of flow injection and syringe pump to optimize the mass spectrometry parameters of bile acids, including the parent ion Q1, fragment ion Q3, declustering voltage DP, and collision energy CE, to construct a bile acid database. S12: Optimize the chromatographic conditions. S13: Identify substances that often cause poor detection or inaccurate quantification due to overloaded mass spectrometry response, perform negative optimization on the mass spectrometry parameters DP and CE of their compounds to reduce their mass spectrometry response, and add the negatively optimized bile acid ion pair information to the bile acid database. After the biological sample is analyzed on the machine, different quantitative ion pairs are selected according to the content of bile acids in the biological sample during data processing. Among them, different quantitative ion pairs refer to the original quantitative ion pairs or negatively optimized ion pairs. When the bile acid response exceeds the upper limit of mass spectrometry, the negatively optimized ion pairs are selected; when it does not exceed the limit, the original quantitative ion pairs are selected.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows.

[0007] Further, the liquid chromatography is ultra-high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is an ACQUITY HSST3 column, i.d. 2.1×100 mm, 1.8 μm; the column temperature is 40 °C; the flow rate is 0.35 mL / min; the injection volume is 3 μL; the gradient elution program: at 0 min, A / B is 95:5 (V / V), at 1 min, A / B is 60:40 (V / V), at 7 min, it is 50:50 (V / V), at 12 min, it is 25:75 (V / V), at 14 min, it is 5:95 (V / V), at 16.0 min, it is 95:5 (V / V), and at 17.5 min, it is 95:5 (V / V).

[0008] Further, the mass spectrometry conditions are as follows: the ESI source negative ion mode is used for detection, and the scanning mode is the multiple reaction monitoring mode; the spray voltage is -4500 V, the ion source temperature is 550 °C, and the collision gas is 7 psi; the curtain gas is 35 psi, and the nebulizing gas and auxiliary gas are 50 psi and 60 psi respectively.

[0009] Further, the optimized chromatographic conditions of S12 include the optimization of the mobile phase composition, additives, mobile phase elution gradient, and analysis time, enabling baseline separation of isomers. The mobile phase is at least one of methanol, acetonitrile, and water, and the additives are 2 - 10 mM ammonium acetate, formic acid with a volume concentration of 0.1 - 0.01%, and acetic acid solution with a volume concentration of 0.1 - 0.01%.

[0010] Further, it also includes step S5 for the qualitative and quantitative analysis of bile acids; Step S2: Pipette or weigh a biological sample, add the isotope internal standard of the substance to be detected, add different extraction solutions respectively, vortex evenly, and centrifuge to obtain the sample extraction solution. After the sample extraction solution is subjected to on-machine detection, select the optimal extraction conditions according to the peak shape and resolution; Step S3 includes: taking a 60 - bile acid mixed standard, diluting it into a series of standard curve concentration points with the extraction solution, and each concentration point contains an equal amount of isotope internal standard; Step S4: Use the optimal extraction solution in step S2 and the standard curve concentration points in step S3, and perform on-machine detection and analysis using the established LC-MS / MS method. Different sample types select different quantitative standard curves according to the mass spectrometry response of the actual bile acids for data processing and quantification.

[0011] Further, the mobile phase composition includes mobile phase A and mobile phase B. Mobile phase A is water, containing 5 mM ammonium acetate and formic acid solution with a volume concentration of 0.01%; mobile phase B is acetonitrile, containing formic acid solution with a volume concentration of 0.01%.

[0012] Further, the extraction solution is a methanol solution, an acetonitrile solution, or a methanol-acetonitrile mixed solution with a volume percentage of 20%.

[0013] Further, the extraction conditions for bile acids in different biological samples are as follows: Blood sample: Take 50 μL of blood sample and add 200 μL of a methanol-acetonitrile mixed solution with a volume percentage of 20% containing an internal standard extraction solution. Bile sample: Take 20 μL of bile sample and add 30 μL of a methanol-acetonitrile mixed solution with a volume percentage of 20% and 200 μL of an internal standard extraction solution. Tissue sample: Take 20 mg of tissue sample and add 200 μL of a methanol-acetonitrile mixed solution with a volume percentage of 20% containing an internal standard extraction solution. Fecal sample: Take 20 mg of fecal sample and add 500 μL of a methanol-acetonitrile mixed solution with a volume percentage of 20% containing an internal standard extraction solution. After selecting different above-mentioned extraction conditions for different sample types, vortex for 5 min, centrifuge at 12,000 r / min for 15 min at 4°C, and take the supernatant for instrumental analysis after centrifugation.

[0014] Further, there are 60 kinds of bile acids, including lithocholic acid, allolithocholic acid, 23-nor-deoxycholic acid, 3,7-diketocholic acid, 3,6-diketocholic acid, 5-β-cholanic acid-3α-ol-6-one, 7-ketolithocholic acid, 12-ketolithocholic acid, isolithocholic acid, hyodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, 3β-dehydrocholic acid, murideoxycholic acid, 3β-ursodeoxycholic acid, 3β-deoxycholic acid, norcholic acid, dehydrocholic acid, 7,12-diketolithocholic acid, 6,7-diketolithocholic acid, 7-ketodeoxycholic acid, 12-oxo-chenodeoxycholic acid, 12-dehydrocholic acid, β-muricholic acid, ursolic acid, ω-muricholic acid, 3β-cholic acid, hyocholic acid, cholic acid, allocholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycoursodeoxycholic acid, glycochenodeoxycholic acid, lithocholic acid-3-sulfate, glycine dehydrocholic acid, glycine cholic acid, glycine hyocholic acid, 3β-glycocholic acid, chenodeoxycholic acid 3-sulfate disodium salt, deoxycholic acid 3-O-sulfate disodium salt, ursodeoxycholic acid-3-sulfate, taurolithocholic acid, cholic acid 7-sulfate, cholic acid 3-sulfate sodium salt, taurodeoxycholic acid sodium salt, tauroursodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid-3-sulfate, taurocholic acid, tauro-β-muricholic acid, taurohyocholic acid, glycochenodeoxycholic acid 3-sulfate disodium salt, glycoursodeoxycholic acid 3-sulfate sodium salt, taurolithocholic acid-3-sulfate, chenodeoxycholic acid 24-acyl-β-D-glucuronide, taurocholic acid 3-sulfate sodium salt.

[0015] Further, there are 16 substances that often cause poor detection or inaccurate quantification due to mass spectrometry response overload, namely CA-7S, β-MCA, 7-KDCA, HDCA, GLCA, GCDCA, GUDCA, GCA, GDCA, TUDCA, THDCA, GHDCA, GCDCA-3S, TLCA, TDCA, and TCA in bile and feces.

[0016] Further, for the 16 bile acids, the DPs of the original quantitative ion pairs are -50, -40, -40, -45, -22, -30, -20, -30, -54, -45, -50, -50, -50, -26, -45, -36 respectively, and the CEs are -12, -20, -15, -15, -40, -40, -40, -50, -40, -90, -12, -12, -12, -90, -90, -90 respectively; the DPs of the ion pairs after negative optimization are -100, -100, -100, -120, -100, -80, -100, -120, -80, -100, -100, -80, -100, -80, -80, -100 respectively, and the CEs are -50, -50, -40, -60, -79, -84, -87, -76, -77, -143, -60, -50, -40, -142, -144, -163 respectively.

[0017] Further, it also includes step S5 for the qualitative and quantitative analysis of bile acids. Step S5 includes: S51. Using the constructed database, perform LC-MS / MS analysis on the standard solutions of bile acids and their isotope internal standards to obtain the retention times of different bile acids, and qualitatively analyze the bile acids in biological samples according to the characteristic fragments and retention times of different bile acids; S52. Prepare standard samples containing various bile acids at different concentrations for LC-MS / MS analysis. According to the different mass spectrometry peak area responses corresponding to different concentrations, establish a linear curve, and substitute the peak area responses of each bile acid in different sample types into the linear curve to obtain the contents of each bile acid in different sample types.

[0018] Further, in step S3: Take a certain amount of the 60-bile acid mixed standard, and dilute it with the extraction solution into a series of calibration curve concentration points of 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400 ng / mL. Each concentration point contains an equal amount of isotope internal standard.

[0019] Further, the extraction solution is a methanol-acetonitrile mixed solution with a volume percentage of 20% of the extraction solution containing internal standard. Further, the mobile phase A is ultrapure water, containing 5 mM ammonium acetate and a formic acid solution with a volume concentration of 0.01%; the mobile phase B is acetonitrile, containing a formic acid solution with a volume concentration of 0.01%.

[0020] Further, the tissue sample can be liver, kidney, muscle or pancreatic tissue, preferably liver tissue.

[0021] Further, this method can detect 60 bile acids, can achieve baseline separation of a total of 49 isomers in 14 groups, and the analysis time for one sample is 17.5 min.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects: Compared with the prior art which only detects bile acids in blood, the LC-MS / MS method of the present invention can quantitatively detect 60 bile acids in common biological samples such as blood, tissue, bile, and feces. It covers a complete range of biological sample types and has a wide application range, which is of great significance for the screening, diagnosis and differentiation of intestinal diseases; By adjusting the mobile phase composition, additive dosage, gradient ratio, and analysis time, the present invention can achieve baseline separation of a total of 49 isomers in 14 groups, and the analysis time for one sample is 17.5 min; compared with the patent text with the publication number CN110596295A and the title "A method for detecting bile acids" which detects 38 bile acids and has an analysis time of 26 min, and the patent text with the publication number CN113533566A and the title "A method for rapidly determining the content of 41 bile acids in serum based on LC-MS / MS method" which detects 41 bile acids and has an analysis time of 38 min, it has the advantages of detecting a large number of bile acids, covering all bile acids, good separation performance, and short analysis time; For the 16 bile acids that often reach the upper limit of the mass spectrometry response in bile and feces, the present invention performs negative optimization of the mass spectrometry parameters. The detection method simultaneously retains the original quantitative ion pair and the negatively optimized quantitative ion pair, which can achieve the effect of simultaneously quantifying high-concentration and low-concentration bile acids in one detection, breaking the traditional thinking that the sample content is too high and exceeds the mass spectrometry upper limit, requiring one or more dilutions and multiple machine detections, and greatly saving the time cost of extraction and machine detection.

[0023] The pretreatment of the present invention is simple, can achieve rapid extraction of various bile acids without affecting the sensitivity of the method, has low cost, high efficiency, and is extremely easy to automate. Description of the Drawings

[0024] Figure 1 It is an LC-MS / MS detection chart of a related bile acid standard product containing 60 bile acids mentioned in the present invention, and different colors represent different bile acids; Figure 2 LC-MS / MS detection chart of taurocholic acid-related isomer standards. Different retention times (RT) represent different substances; Figure 3 The upper and lower two figures are the detection charts of TCA (taurocholic acid) in mouse fecal samples under the "original and negative optimized" mass spectrometry parameters. The lower figure is the detection chart of negative optimization; Figure 4 The upper and lower two figures are the detection charts of HDCA (hyodeoxycholic acid) in mouse bile samples under the "original and negative optimized" mass spectrometry parameters. The lower figure is the detection chart of negative optimization; Figure 5 It is a stability superimposed chart of continuously detecting bile acid mixed standards for 72 hours. Detailed implementation manners

[0025] For the convenience of understanding this application, the following will provide a more comprehensive description of this application. This application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of this application more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0027] Aiming at the deficiencies of the prior art, the present invention uses the LC-MS / MS method to quantitatively detect 60 bile acids in common biological samples such as blood, liver tissue, bile, and feces. The mobile phase components and composition of this method have been improved, and baseline separation of 49 bile acid isomers in 14 groups can be achieved. It has the advantages of simple pretreatment (without complex pretreatment steps such as derivatization and solid-phase extraction), a large number of detected bile acids, full coverage of biological sample types, good separation performance, short analysis time, etc. Moreover, for 16 bile acids with relatively high contents in bile and feces and often reaching the upper limit of mass spectrometry response, negative optimization of mass spectrometry parameters has been carried out. Both the original quantitative ion pairs and the negatively optimized quantitative ion pairs are retained in the detection method, and the effect of simultaneously quantifying high-concentration and low-concentration bile acids can be achieved in one detection, breaking the traditional thinking that the sample content is too high and exceeds the upper limit of mass spectrometry, requiring one or multiple dilutions and multiple machine detections. It has the advantages of convenient operation and more accurate and reliable quantitative results.

[0028] The blood samples in the embodiments of the present invention are derived from mouse serum and plasma, the bile samples are derived from mouse bile, the tissue samples are derived from mouse liver and kidney, and the fecal samples are derived from mouse feces. Female C57BL / 6 healthy mice, 6 weeks old, with a body weight of 20 - 22 g.

[0029] An LC-MS / MS method for detecting bile acids in biological samples, which is detected by liquid chromatography-tandem mass spectrometry, and includes the following steps: S1, construction of a database, separation of isomers, and negative optimization of bile or fecal overload substances; S2, optimization of the extraction conditions of biological samples; S3, preparation of a standard curve; S4, chromatographic and mass spectrometric detection and analysis. The biological samples are blood samples, bile samples, tissue samples or fecal samples. Step S1 includes: S11, taking bile acid standards, preparing a stock solution of the standards, and optimizing the mass spectrometry parameters of bile acids by means of flow injection with a syringe pump, including precursor ion Q1, fragment ion Q3, declustering potential DP, and collision energy CE, to construct a bile acid database; S12, optimizing the chromatographic conditions; S13, determining substances that often cause poor detection or inaccurate quantification due to mass spectrometry response overload, negatively optimizing the mass spectrometry parameters DP and CE of their compounds to reduce their mass spectrometry response, and adding the negatively optimized bile acid ion pair information to the bile acid database. After the biological samples are loaded onto the machine for detection, different quantitative ion pairs are selected according to the content of bile acids in the biological samples during data processing; Among them, the different quantitative ion pairs refer to the original quantitative ion pairs or negatively optimized ion pairs. When the bile acid response exceeds the mass spectrometry upper limit, the negatively optimized ion pairs are selected; when it does not exceed the limit, the original quantitative ion pairs are selected.

[0030] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0031] Further, the liquid chromatography is ultra-high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is an ACQUITY HSST3 column, i.d. 2.1×100 mm, 1.8 μm; the column temperature is 40°C; the flow rate is 0.35 mL / min; the injection volume is 3 μL; the gradient elution program: at 0 min, A / B is 95:5 (V / V), at 1 min, A / B is 60:40 (V / V), at 7 min, it is 50:50 (V / V), at 12 min, it is 25:75 (V / V), at 14 min, it is 5:95 (V / V), at 16.0 min, it is 95:5 (V / V), and at 17.5 min, it is 95:5 (V / V).

[0032] Furthermore, the mass spectrometry conditions are as follows: ESI source negative ion mode is used for detection, and the scanning mode is multiple reaction monitoring mode; the spray voltage is -4500 V, the ion source temperature is 550 °C, and the collision gas is 7 psi; the curtain gas is 35 psi, and the nebulizing gas and auxiliary gas are 50 psi and 60 psi respectively.

[0033] Furthermore, the optimized chromatographic conditions of S12 include the optimization of mobile phase composition, additives, mobile phase elution gradient and analysis time, so that isomers can be baseline separated. The mobile phase is at least one of methanol, acetonitrile and water, and the additives are 2 - 10 mM ammonium acetate, formic acid with a volume concentration of 0.1 - 0.01%, and acetic acid solution with a volume concentration of 0.1 - 0.01%.

[0034] Furthermore, it also includes step S5 for the qualitative and quantitative analysis of bile acids; Step S2: Pipette or weigh a biological sample, add an isotope internal standard of the substance to be detected, add different extraction solutions respectively, vortex evenly, centrifuge to obtain a sample extract. After the sample extract is analyzed by machine, select the optimal extraction conditions according to the peak shape and resolution; Step S3 includes: taking a 60 - bile acid mixed standard, diluting it with an extraction solution into a series of calibration curve concentration points, and each concentration point contains an equal amount of isotope internal standard; Step S4: Use the optimal extraction solution in step S2 and the calibration curve concentration points in step S3, and perform on - machine detection and analysis using the established LC - MS / MS method. Different sample types select different quantitative calibration curves for data processing and quantification according to the mass spectrometry response of the actual bile acids.

[0035] Furthermore, the mobile phase composition includes mobile phase A and mobile phase B. Mobile phase A is water, containing 5 mM ammonium acetate and formic acid solution with a volume concentration of 0.01%; mobile phase B is acetonitrile, containing formic acid solution with a volume concentration of 0.01%.

[0036] Furthermore, the extraction solution is a methanol solution, an acetonitrile solution, or a methanol - acetonitrile mixed solution with a volume percentage of 20%.

[0037] Furthermore, the extraction conditions for bile acids in different biological samples: Blood sample: Take 50 μL of blood sample, add 200 μL of methanol - acetonitrile mixed solution with a volume percentage of 20% containing internal standard extraction solution; Bile sample: Take 20 μL of bile sample, add 30 μL of methanol - acetonitrile mixed solution with a volume percentage of 20% and 200 μL of internal standard extraction solution; Tissue sample: Take 20 mg of tissue sample, add 200 μL of methanol - acetonitrile mixed solution with a volume percentage of 20% containing internal standard extraction solution; Fecal sample: Take 20 mg of fecal sample and add 500 μL of an internal standard extraction solution of a methanol-acetonitrile mixed solution with a volume percentage of 20%. After selecting different above extraction conditions for different sample types, vortex for 5 min, centrifuge at 12,000 r / min for 15 min at 4°C, and take the supernatant for instrumental analysis after centrifugation.

[0038] Furthermore, there are 60 kinds of bile acids, including lithocholic acid, allolithocholic acid, 23-nor-deoxycholic acid, 3,7-diketocholic acid, 3,6-diketocholic acid, 5-β-cholanic acid-3α-ol-6-one, 7-ketolithocholic acid, 12-ketolithocholic acid, isolithocholic acid, hyodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, 3β-dehydrocholic acid, murideoxycholic acid, 3β-ursodeoxycholic acid, 3β-deoxycholic acid, norcholic acid, dehydrocholic acid, 7,12-diketolithocholic acid, 6,7-diketolithocholic acid, 7-ketodeoxycholic acid, 12-oxo-chenodeoxycholic acid, 12-dehydrocholic acid, β-muricholic acid, ursolithocholic acid, ω-muricholic acid, 3β-cholic acid, hyocholic acid, cholic acid, allocholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycoursodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, lithocholic acid-3-sulfate, glycodehydrocholic acid, glycocholic acid, glycoursodeoxycholic acid, 3β-glycocholic acid, chenodeoxycholic acid 3-sulfate disodium salt, deoxycholic acid 3-O-sulfate disodium salt, ursodeoxycholic acid-3-sulfate, taurolithocholic acid, cholic acid 7-sulfate, cholic acid 3-sulfate sodium salt, taurodeoxycholic acid sodium salt, tauroursodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid-3-sulfate, taurocholic acid, tauro-β-muricholic acid, taurohyocholic acid, glycochenodeoxycholic acid 3-sulfate disodium salt, glycoursodeoxycholic acid 3-sulfate sodium salt, taurolithocholic acid-3-sulfate, chenodeoxycholic acid 24-acyl-β-D-glucuronide, taurocholic acid 3-sulfate sodium salt.

[0039] Furthermore, there are 16 substances that often cause poor detection or inaccurate quantification due to mass spectrometry response overload, namely CA-7S, β-MCA, 7-KDCA, HDCA, GLCA, GCDCA, GUDCA, GCA, GDCA, TUDCA, THDCA, GHDCA, GCDCA-3S, TLCA, TDCA, TCA in bile and feces.

[0040] Further, for the 16 bile acids, the DPs of the original quantitative ion pairs were -50, -40, -40, -45, -22, -30, -20, -30, -54, -45, -50, -50, -50, -26, -45, -36 respectively, and the CEs were -12, -20, -15, -15, -40, -40, -40, -50, -40, -90, -12, -12, -12, -90, -90, -90 respectively. After negative optimization, the DPs of the ion pairs were -100, -100, -100, -120, -100, -80, -100, -120, -80, -100, -100, -80, -100, -80, -80, -100 respectively, and the CEs were -50, -50, -40, -60, -79, -84, -87, -76, -77, -143, -60, -50, -40, -142, -144, -163 respectively.

[0041] Further, it also includes step S5 for the qualitative and quantitative analysis of bile acids. Step S5 includes: S51. Using the constructed database, perform LC-MS / MS analysis on the standard solutions of bile acids and their isotope internal standards to obtain the retention times of different bile acids, and qualitatively analyze the bile acids in biological samples based on the characteristic fragments and retention times of different bile acids. S52. Prepare standard samples containing various bile acids at different concentrations for LC-MS / MS analysis. According to the different corresponding mass spectrometry peak area responses at different concentrations, establish a linear curve, and substitute the peak area responses of each bile acid in different sample types into the linear curve to obtain the contents of each bile acid in different sample types.

[0042] Further, in step S3: Take a certain amount of the 60-bile acid mixed standard, and dilute it with the extraction solution into a series of calibration curve concentration points of 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400 ng / mL, and each concentration point contains an equal amount of isotope internal standard.

[0043] Further, the extraction solution is a methanol-acetonitrile mixed solution with a volume percentage of 20% of the internal standard-containing extraction solution. Further, the mobile phase A is ultrapure water containing 5 mM ammonium acetate and a formic acid solution with a volume concentration of 0.01%; the mobile phase B is acetonitrile containing a formic acid solution with a volume concentration of 0.01%.

[0044] Further, the tissue sample can be liver, kidney, muscle or pancreatic tissue, preferably liver tissue.

[0045] Furthermore, this method can detect 60 bile acids, achieve baseline separation of 49 isomers in 14 groups, and the analysis time for one sample is 17.5 min.

[0046] Example 1 The technical solution of the present invention mainly includes the following content: Construction of the S1 database, separation of isomers, and negative optimization of overloaded substances in bile or feces The establishment of the 60-bile-acid database in this method and the negative optimization of overloaded substances in feces and bile include the following steps: S11. Purchase 60 commercially available bile acid standards, prepare the standard stock solution, and precisely optimize the mass spectrometry parameters of the 60 bile acids by means of flow injection using a syringe pump, including the optimal parent ion Q1, fragment ion Q3, declustering voltage DP, and collision energy CE, to construct the bile acid database; S12. Optimize the chromatographic conditions, including the composition of the mobile phase (methanol, acetonitrile, or water), additives (2 - 10 mM ammonium acetate, formic acid solution with a volume concentration of 0.1 - 0.01%, acetic acid solution with a volume concentration of 0.1 - 0.01%), the elution gradient of the mobile phase (gradient ratio delay, promoting separation, etc.), and the analysis time, etc., so that 49 isomers in 14 groups can achieve baseline separation. The optimal mobile phase composition and additives are as follows: mobile phase A is ultrapure water, containing 5 mM ammonium acetate and formic acid solution with a volume concentration of 0.01%; mobile phase B is acetonitrile, containing formic acid solution with a volume concentration of 0.01%. The optimal elution gradient of the mobile phase is 0 min, A / B is 95:5 (V / V); 1 min, A / B is 60:40 (V / V); 7 min is 50:50 (V / V); 12 min is 25:75 (V / V); 14 min is 5:95 (V / V); 16.0 min is 95:5 (V / V); 17.5 min is 95:5 (V / V).

[0047] Figure 1 It is the LC-MS / MS detection chart of the related bile acid standards containing the 60 bile acids mentioned in the present invention. The analysis time is 17.5 min (the time in the chart needs to add the equilibration time of 1.5 min). Different colors represent different substances, at least the 60 bile acids mentioned in the present invention. Figure 2 It is the LC-MS / MS detection chart of the related isomer standards of taurocholic acid. The analysis time is 17.5 min (the time in the chart needs to add the equilibration time of 1.5 min). Different times represent different substances. From Figure 1 and Figure 2 it can be seen that the 60 bile acids are all well separated under the 17.5-min gradient, especially for the isomers, which can be well separated.

[0048] S13. Summarize the project experience of fecal and bile sample types, identify substances that often cause poor detection or inaccurate quantification due to mass spectrometry response overload, perform negative optimization on the compound mass spectrometry parameters DP and CE for these substances to reduce their mass spectrometry response, and add the information of the 16 ion pairs of bile acids after negative optimization to the bile acid database. After the biological samples are tested on the machine, only different quantitative ion pairs need to be selected according to the content of bile acids in the biological samples during data processing (for example, when the response of bile acids in bile and feces exceeds the mass spectrometry upper limit, select the negatively optimized ion pairs; when it does not exceed the limit, use the original quantitative ion pairs), without multiple dilutions of the samples and multiple tests on the machine, which greatly saves the time cost of extraction and testing on the machine. Attached figure Figure 3 and Figure 4 show the detection spectra of feces and bile under different mass spectrometry parameters. From Figure 3 and Figure 4 it can be seen that after negative optimization, the mass spectrometry responses of TCA (taurocholic acid) in feces and HDCA (hyodeoxycholic acid) in bile decreased significantly, and the chromatographic peak shape improved from "flat peak" to "symmetric chromatogram".

[0049] S2 Optimization of extraction conditions for biological samples S3 Preparation of standard curve S4 Chromatographic and mass spectrometric detection and analysis The above extraction solution and standard curve concentration points were analyzed by LC-MS / MS method that has been constructed on the machine. For different sample types such as blood and tissue, bile and feces, different quantitative standard curves were selected according to the actual mass spectrometry response of bile acids for data processing and quantification.

[0050] Example 2 Detection of bile acids in biological samples The optimal extraction conditions for bile acids in different biological samples are as follows: (1) Take 50 μL of blood sample and add 200 μL of internal standard extraction solution containing a 20% (v / v) methanol-acetonitrile mixed solution; (2) Take 20 μL of bile sample and add 30 μL of a 20% (v / v) methanol-acetonitrile mixed solution and 200 μL of internal standard extraction solution; (3) Take 20 mg of tissue sample and add 200 μL of internal standard extraction solution containing a 20% (v / v) methanol-acetonitrile mixed solution; (4) Take 20 mg of fecal sample and add 500 μL of internal standard extraction solution containing a 20% (v / v) methanol-acetonitrile mixed solution; After selecting different above extraction solutions for different sample types, vortex for 5 min, centrifuge at 12,000 r / min for 15 min at 4 °C, and take the supernatant for analysis on the machine after centrifugation.

[0051] Chromatographic and mass spectrometric detection: Instrument parameter conditions: The data acquisition instrument system used for LC-MS / MS detection in the present invention mainly includes an ultra-high performance liquid chromatography (Waters HClass) and a tandem mass spectrometer MS / MS (Applied Biosystems 6500 Quadrupole Trap).

[0052] (1) The ultra-high performance liquid chromatography conditions mainly include: the chromatographic column is an ACQUITY HSS T3 column, i.d. 2.1×100 mm, 1.8 μm; mobile phase A is ultrapure water (containing 5 mM ammonium acetate and a formic acid solution with a volume concentration of 0.01%); mobile phase B is acetonitrile (containing a formic acid solution with a volume concentration of 0.01%); the column temperature is 40°C; the flow rate is 0.35 mL / min; the injection volume is 3 μL; gradient elution program: at 0 min, A / B is 95:5 (V / V), at 1 min, A / B is 60:40 (V / V), at 7 min, it is 50:50 (V / V), at 12 min, it is 25:75 (V / V), at 14 min, it is 5:95 (V / V), at 16.0 min, it is 95:5 (V / V), at 17.5 min, it is 95:5 (V / V).

[0053] (2) Mass spectrometry conditions: Detection is carried out in the negative ion mode of the ESI source, and the scanning mode is the multiple reaction monitoring mode (MRM); the spray voltage is -4500 V, the ion source temperature is 550°C, the collision gas is 7 psi; the curtain gas is 35 psi, and the nebulizing gas and auxiliary gas are 50 psi and 60 psi respectively.

[0054] Qualitative and quantitative analysis of bile acids: (1) Using the constructed database, standard solutions of 60 bile acids and their isotope internal standards are analyzed by LC-MS / MS under the same operating conditions as above to obtain the retention times of different bile acids. Different bile acids in biological samples are qualitatively analyzed based on the characteristic fragments and retention times of different bile acids, as shown in Table 1; (2) Standard samples containing 60 bile acids with different concentrations are prepared for LC-MS / MS analysis. According to the different mass spectrometry peak area responses corresponding to different concentrations, a linear curve is established. By substituting the peak area responses of each bile acid in different sample types into the linear curve, the contents of each bile acid in different sample types can be obtained, Figure 5 demonstrating the stability of the continuous detection method for bile acids, as shown in Table 2; Table 1 Optimal quantitative ion pairs and isomer groupings of different bile acids

[0055] Table 2 Calibration Curve: weighting is set to 1 / x

[0056] Note: The bile acid with the suffix "_1" in the abbreviation refers to the negative optimization of this bile acid in bile and feces.

[0057] Table 3 Spike Recovery and Precision in Biological Samples

[0058] As can be seen from Table 3, for the 60 metabolites detected by the method of the present invention in different biological samples, the recovery rates can reach 70 - 130%, and more than 90% of the substances have intra-day precision and inter-day precision for 3 consecutive days less than 15%, indicating that the accuracy and stability of the method of the present invention are very good.

[0059] Advantages of the present invention: Compared with the prior art that only detects bile acids in blood, the present invention uses LC-MS / MS method to quantitatively detect 60 bile acids in blood, tissue, bile and feces in common biological samples, covering a complete range of biological sample types and having a wide application range, which is of great significance for the screening, diagnosis and differentiation of intestinal diseases; By adjusting the mobile phase composition, additive dosage, gradient ratio and analysis duration, the present invention can achieve baseline separation of 49 isomers in 14 groups, and the analysis duration of one sample is 17.5 min; compared with the patent text with the publication number CN110596295A and the title "A Method for Detecting Bile Acids" that detects 38 bile acids and has an analysis duration of 26 min, and the patent text with the publication number CN113533566A and the title "A Method for Rapidly Determining the Content of 41 Bile Acids in Serum Based on LC-MS / MS Method" that detects 41 bile acids and has an analysis duration of 38 min, it has the advantages of detecting more types of bile acids, covering all bile acids, good separation performance and short analysis time; For the 16 bile acids that often reach the upper limit of the mass spectrometry response in bile and feces, the present invention performs negative optimization of the mass spectrometry parameters. The detection method retains both the original quantitative ion pair and the negatively optimized quantitative ion pair, which can achieve the effect of simultaneously detecting and quantitatively analyzing high-concentration and low-concentration bile acids in one test, breaking the traditional thinking that when the sample content is too high and exceeds the mass spectrometry upper limit, it needs to be diluted once or multiple times and requires multiple machine detections, greatly saving the time cost of extraction and machine detection.

[0060] The pretreatment of the present invention is simple, and it can realize the rapid extraction of various bile acids without affecting the sensitivity of the method. It has low cost, high efficiency and is very easy to realize automation.

[0061] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A LC-MS / MS method for detecting bile acids in biological samples, characterized in that: The method adopts liquid chromatography-tandem mass spectrometry detection, including the following steps: S1 construction of a database, separation of isomers, and negative optimization of bile or feces overload substances, S2 optimization of biological sample extraction conditions, S3 preparation of a standard curve, and S4 chromatography-mass spectrometry detection and analysis, wherein the biological sample is a blood sample, a bile sample, a tissue sample, or a feces sample; Step S1 comprises: S11, taking bile acid standards, preparing standard stock solutions, and optimizing the mass spectrometry parameters of bile acid using a flow injection needle pump, including parent ion Q1, fragment ion Q3, declustering voltage DP, and collision energy CE, to construct a bile acid database; S12, optimize chromatographic conditions; S13, determining substances that often have poor detection or inaccurate quantification due to mass spectrometry response overload, negatively optimizing the mass spectrometry parameters DP and CE of the compounds to reduce their mass spectrometry response, and adding the negatively optimized bile acid ion pair information to the bile acid database, and after the biological samples are tested on the machine, selecting different quantitative ion pairs according to the content of bile acid in the biological samples during data processing; The different quantitative ion pairs refer to the original quantitative ion pairs or the negative optimized ion pairs. When the bile acid response exceeds the upper limit of the mass spectrum, the negative optimized ion pair is selected, and when it does not exceed the upper limit, the original quantitative ion pair is selected.

2. The LC-MS / MS method for detecting bile acids in a biological sample according to claim 1, characterized in that: The liquid chromatography is an ultra-high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is an ACQUITY HSS T3 column, id2.1×100mm, 1.8 μm; the column temperature is 40°C; the flow rate is 0.35 mL / min; the injection volume is 3 μL; the gradient elution program: 0 min A / B is 95:5 (V / V), 1 min A / B is 60:40 (V / V), 7 min is 50:50 (V / V), 12 min is 25:75 (V / V), 14 min is 5:95 (V / V), 16.0 min is 95:5 (V / V), and 17.5 min is 95:5 (V / V).

3. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 1, characterized in that: Mass spectrometry conditions: ESI source negative ion mode detection, scanning mode is multiple reaction monitoring mode; spray voltage is -4500V, ion source temperature is 550℃, collision gas is 7psi; curtain gas is 35psi, nebulizer and auxiliary gas are 50psi and 60psi respectively.

4. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 1, characterized in that: The S12 optimized chromatographic conditions include optimization of mobile phase composition, additives, mobile phase elution gradient and analysis time, so that isomers can be baseline separated, the mobile phase is at least one of methanol, acetonitrile and water, and the additives are 2-10 mM ammonium acetate, formic acid with a volume concentration of 0.1-0.01%, and acetic acid solution with a volume concentration of 0.1-0.01%.

5. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 1, characterized in that: Step S2: Pipette or weigh the biological sample, add the isotope internal standard of the substance to be tested, add different extracts respectively, vortex evenly, centrifuge to obtain the sample extract, and after the sample extract is tested on the machine, select the optimal extraction conditions according to the peak shape and separation degree; Step S3 comprises: taking 60 bile acid mixed standards, and diluting them into a series of standard concentration points using an extract, each concentration point containing an equal amount of isotope internal standard; Step S4: using the optimal extract in step S2 and the calibration curve concentration point in step S3, using the established LC-MS / MS method to perform on-machine detection and analysis, different sample types select different quantitative calibration curves for data processing and quantification according to the mass spectrometry response of actual bile acids; The process also includes step S5 of bile acid qualitative and quantitative analysis.

6. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 4, characterized in that: The mobile phase composition includes mobile phase A and mobile phase B, wherein the mobile phase A is water, containing 5 mM ammonium acetate and a formic acid solution with a volume concentration of 0.01%; and the mobile phase B is acetonitrile, containing a formic acid solution with a volume concentration of 0.01%.

7. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 5, characterized in that: The extracting solution is a methanol solution, an acetonitrile solution, or a methanol-acetonitrile mixed solution with a volume percentage of 20%.

8. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 7, characterized in that: Extraction conditions of bile acids from different biological samples: Blood sample: Take 50 μL of blood sample and add 200 μL of 20% by volume methanol-acetonitrile mixed solution containing internal standard extract; Bile sample: Take 20 μL of bile sample, add 30 μL of 20% methanol-acetonitrile mixed solution and 200 μL of internal standard extract; Tissue sample: Take 20 mg of tissue sample and add 200 μL of 20% by volume methanol-acetonitrile mixed solution containing internal standard extract; Fecal sample: Take 20 mg of fecal sample and add 500 μL of 20% by volume methanol-acetonitrile mixed solution containing internal standard extract; After selecting different extraction conditions for different sample types, the samples were vortexed for 5 min and centrifuged at 12,000 r / min for 15 min at 4°C. The supernatant was taken for analysis.

9. The LC-MS / MS method for detecting bile acid in a biological sample according to claim 1, characterized in that: There are 60 kinds of bile acids, including lithocholic acid, allocholic acid, 23-demethyldeoxycholic acid, 3,7-diketocholic acid, 3,6-diketocholic acid, 5-β-cholic acid-3α-ol-6-one, 7-ketolithocholic acid, 12-ketolithocholic acid, isochenodeoxycholic acid, hyodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, 3β-dehydrocholic acid, murideoxycholic acid , 3β-ursodeoxycholic acid, 3β-deoxycholic acid, norcholic acid, dehydrocholic acid, 7,12-diketolithocholic acid, 6,7-diketolithocholic acid, 7-ketodeoxycholic acid, 12-oxochenodeoxycholic acid, 12-dehydrocholic acid, β-muricolic acid, ursocholic acid, ω-muricolic acid, 3β-cholic acid, hyocholic acid, cholic acid, allocholic acid, glycolithocholic acid, glycochenodeoxycholic acid, Glycoursodeoxycholic acid, glycodeoxycholic acid, glycohyodeoxycholic acid, lithocholic acid-3-sulfate, glycodehydrocholic acid, glycocholic acid, glycohyodeoxycholic acid, 3β-glycocholic acid, chenodeoxycholic acid 3-sulfate disodium salt, deoxycholic acid 3-O-sulfate disodium salt, ursodeoxycholic acid-3 sulfate, taurolithocholic acid, cholic acid 7 sulfate, cholic acid 3-sulfate sodium salt, tauroyldeoxycholic acid sodium salt, tauroursodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, glycolithocholic acid-3-sulfate, taurocholic acid, tauroβ-muricholic acid, taurohyodeoxycholic acid, glycochenodeoxycholic acid 3 sulfate disodium salt, glycoursodeoxycholic acid 3 sulfate sodium salt, taurolithocholic acid-3-sulfate, chenodeoxycholic acid 24-acyl-bD-glucuronic acid, taurocholic acid 3 sulfate sodium salt.

10. The LC-MS / MS method for detecting bile acids in a biological sample according to claim 1, characterized in that: There are 16 substances that often have poor detection or inaccurate quantification due to mass spectrometry response overload, including CA-7S, β-MCA, 7-KDCA, HDCA, GLCA, GCDCA, GUDCA, GCA, GDCA, TUDCA, THDCA, GHDCA, GCDCA-3S, TLCA, TDCA, and TCA in bile and feces.

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