CN114509513A

CN114509513A - Liquid chromatography high-resolution mass spectrometry qualitative and quantitative detection method for bile acid in multiple tissues

Info

Publication number: CN114509513A
Application number: CN202111636454.4A
Authority: CN
Inventors: 谷旭; 秦玉昌; 薛敏; 贾铭; 韦娴; 刘鸿鹤; 王杰; 任萃文
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-05-17
Anticipated expiration: 2041-12-29
Also published as: CN114509513B

Abstract

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a qualitative and quantitative detection method of bile acid in multiple tissues by liquid chromatography high-resolution mass spectrometry. The detection method comprises the following steps: preparing a mixed internal standard working solution and a standard solution, pretreating a bile acid sample to be detected, and then performing liquid chromatography mass spectrometry, wherein the mobile phase A is 0.05-0.2% formic acid water and the mobile phase B is 0.05-0.2% formic acid acetonitrile in the liquid chromatography conditions, gradient elution is adopted, the flow rate is 350 mul/min, and the column temperature is 35-45 ℃. The mass spectrometry conditions are that an electrospray ion source is adopted, negative ion scanning is carried out, and the pressure of an atomizer is 48-52 psi; collision energy 58-62V; the capillary voltage is 5.2-5.7 kV; the ion temperature is 590-610 ℃; the mass range m/z is 50-800 Da. The invention establishes a qualitative and quantitative detection method for bile acid in various tissues, can realize qualitative and quantitative detection of various bile acid in various animal tissues such as fish, mice and the like or medicinal materials such as arisaema cum bile and the like, and has the advantages of simple method, simple reagent and sensitive detection.

Description

Liquid chromatography high-resolution mass spectrometry qualitative and quantitative detection method for bile acid in multiple tissues

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a qualitative and quantitative detection method of bile acid in multiple tissues by liquid chromatography high-resolution mass spectrometry.

Background

BAs are amphiphilic sterol molecules synthesized from cholesterol in the liver, which are excreted into bile as one of the major components of bile. The BAs (amino acid-acyl-binding primary bile acids, cholic acid [ CA ], chenodeoxycholic acid [ CDCA ] and their secondary metabolites) are secreted by the hepatic parenchymal cells into the tubules, which are the main driving force for bile acid production. The BAs, along with other bile components, all flow into the small intestine, acting to emulsify and absorb fat, cholesterol and fat-soluble vitamins. Upon reaching the terminal ileum, the BAs are almost completely (95%) absorbed by the active absorption mechanism, and the BAs, which are about as they are lost in the stool, are balanced by the daily synthetic BAs. In the distal small intestine and colon, primary BAs (CA and CDCA) are often bacterially de-bound and dehydroxylated to form secondary BAs (i.e., deoxycholic acid [ DCA ] and lithocholic acid [ LCA ]). These secondary BAs can be passively reabsorbed and form part of the total bile acid pool of the hepatic-intestinal circulation.

For many years, it has been believed that the function of BAs has been largely limited to stimulating hepatic bile flow and bile excretion, and to assisting in the digestion and absorption of fat from the intestinal lumen. However, research over the past two decades has recognized that BAs may act as signaling molecules through various receptors to regulate their own synthesis and other metabolic processes such as glucose, lipid, energy homeostasis.

The modulation of BAs is mediated through specific BA-activating receptors, including members of the nuclear receptor superfamily (farnesoid X receptors [ FXR; NR1H4], vitamin D receptors [ N R I1] and pregnane X receptors [ N R I2], and members of the G protein-coupled receptor superfamily (TGR5 and sphingosine-1-phosphate receptor 2). The receptors are expressed by tissues of the gastrointestinal circulation and are not limited to the liver and the gastrointestinal system, where they modulate the systemic action of BAs.

As an endogenous metabolite, bile acids are various in types, show diversified and even opposite biochemical and physiological characteristics due to different side chain structures, stereochemistry properties, number and positions of hydroxyl groups, and the like, at present, related researches are mainly carried out by detecting serum total bile acid levels, and wrong judgment is easily caused due to poor sensitivity and specificity. Therefore, the method can help us to analyze and research the whole physiological and biochemical process of the organism by comprehensively and accurately monitoring the content of various types of bile acid in the biological sample.

The Chinese patent application CN109030676A discloses a tandem mass spectrometry detection kit for simultaneously determining 16 bile acids and application thereof, and the kit comprises a standard substance, a quality control substance, an internal standard substance, a precipitator, a diluent and a mobile phase, wherein the diluent consists of water, methanol, ethanol and acetonitrile, the volume ratio of the water, the methanol, the ethanol and the acetonitrile is 1-50: 0-90: 0-50, and the methanol, the ethanol and the acetonitrile are not 0 at the same time; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is ammonium formate or ammonium acetate-formic acid or acetic acid aqueous solution, and the mobile phase B is methanol and/or acetonitrile. The disadvantages of this method include the following: 1, the pretreatment process is complicated, various reagents are used, and the time is long. 2, the number of bile acids detected is small and some important bile acids are lacking.

The existing qualitative and quantitative method for bile acid mainly has the following defects that on one hand, the types of the quantifiable bile acid are few, the quantifiable bile acid is mainly four kinds of free bile acid, namely Cholic Acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and a small amount of lithocholic acid (LCA), and the used method is basically low-resolution mass spectrum, such as liquid chromatography tandem triple quadrupole mass spectrum and the like; on the other hand, serum is usually selected as a biological sample for the quantification of bile acid, but the methods for extracting and detecting bile acid from other tissue samples such as bile and liver are less involved, the distribution of the bile acid in different tissues is different, the interference of matrixes of different tissues in the analysis and detection process is different, and the blood analysis method cannot cover all the tissues. Due to the continuous discovery of bile acid function, the establishment of a method for simultaneously qualitatively and quantitatively analyzing and detecting multiple kinds of bile acid in each part of organism by high resolution mass spectrum is necessary and urgently needed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention establishes a qualitative and quantitative detection method for bile acid in various tissues, the method can realize qualitative and quantitative detection of various bile acid in various tissues of animals such as fish, mice and the like or medicinal materials such as arisaema cum bile and the like, and the method is simple, the reagent is simple, and the detection is sensitive.

In order to realize the purpose of the invention, the technical scheme is as follows:

a liquid chromatography mass spectrometry detection method of bile acid in multiple tissues comprises performing liquid chromatography mass spectrometry on a bile acid sample to be detected, wherein a mobile phase A in the liquid chromatography condition is 0.05-0.2% formic acid water, a mobile phase B in the liquid chromatography condition is 0.05-0.2% formic acid acetonitrile,

the gradient elution procedure was as follows:

0min，A：B(74-76：24-26，V/V)；

4min，A：B(74-76：24-26，V/V)；

16min，A：B(58-62：38-42，V/V)；

30min，A：B(24-26：74-76，V/V)；

31min，A：B(0-2：98-100，V/V)；

33min，A：B(0-2：98-100，V/V)；

the flow rate is 340-.

Preferably, the conditions of mass spectrometry comprise: adopting an electrospray ion source, scanning negative ions, and controlling the pressure of an atomizer to be 48-52 psi; collision energy 58-62V; the capillary voltage is 5.2-5.7 kV; the ion temperature is 590-610 ℃; the mass range m/z is 50-800 Da.

Preferably, the sample amount in the liquid chromatography condition is 2-5 μ L, the chromatographic column is a carbon eighteen filler chromatographic column, and the carbon eighteen filler chromatographic column is an ACQUITYUPLC BEH C18(1.7 μm, 2.1X 150mm) chromatographic column.

Preferably, the detection method further comprises the preparation of mixed internal standard working solution, and specifically comprises the step of adding methanol into CA-D4, CDCA-D4 and LCA-D4 to prepare the mixed internal standard working solution with the concentration of each component being 4-6 mu g/ml.

Preferably, the detection method further comprises the preparation of a standard solution, specifically comprising: preparing HCA, omega MCA, alpha MCA, T alpha MCA, beta MCA, CDCA, DCA, UDCA, HDCA, 6,7-KLCA, 7,12-KLCA, T beta MCA, T omega MCA, THDCA, TUDCA, GCA, GCDCA, GDCA, GUDCA, LCA, 7-KLCA and 12-KLCA into a standard solution 1 with the concentration of each component being 2-320ng/ml respectively; adding methanol into TCA, TCDCA, CA and TDCA to prepare standard solution 2 with concentration of each component of 10-800 ng/ml.

Preferably, the detection method further comprises a pretreatment step of a bile acid sample to be detected, and when the bile acid sample to be detected is a solid tissue, the pretreatment step comprises: adding the mixed internal standard working solution into the solid tissue, adding 70-80% ethanol solution, crushing, centrifuging, taking supernatant 1, adding glacial acetonitrile, centrifuging for the second time, and taking supernatant 2 to obtain the final product.

Preferably, the mass-to-volume ratio of the solid tissue to the 70-80% ethanol solution is 90-110mg/ml, and the crushing is uniform by intense shaking by using a sample crushing system.

Preferably, the volume ratio of the supernatant 1 to the glacial acetonitrile is 48-52: 48-52.

Preferably, when the bile acid sample to be detected is bile, the pretreatment step includes: adding 75-85% methanol water solution into bile, standing at-18 deg.C to-22 deg.C, centrifuging to obtain supernatant 3, adding 15-45% methanol solution and mixed internal standard working solution, centrifuging again, and collecting supernatant 4.

Preferably, the volume ratio of the bile to the 75-85% methanol water solution is 1: 395-;

preferably, the standing time is 25-35 min.

Preferably, the volume ratio of the supernatant 3 to the 15-45% methanol solution and the mixed internal standard working solution is 38-42:58-62: 1.

Preferably, the temperature of the centrifugation or the secondary centrifugation is 2-5 ℃, the rotating speed is 12000-18000rpm, and the time is 5-15 min.

Preferably, when the bile acid sample to be detected is a plasma sample, the pretreatment step includes: adding the mixed internal standard solution into the plasma, mixing, adding 70-80% ethanol water solution, centrifuging, collecting supernatant, performing C18 solid phase extraction, rinsing with 45-55% methanol solution, discarding, eluting with triethylamine-water-methanol mixed solution system, and collecting eluate.

Preferably, the volume ratio of the blood plasma to the 70-80% ethanol water solution is 1: 8-10.

Preferably, before the extraction, the solid phase of C18 is activated by acetonitrile and 3% formic acid water solution with the mass ratio of 1: 0.8-1.2.

Preferably, the volume ratio of the triethylamine to the water to the methanol is 1-3:16-20: 77-83.

The invention further aims to provide the application of the liquid chromatography-mass spectrometry detection method in qualitative or quantitative detection of various bile acids in animal tissues or traditional Chinese medicines. Compared with the prior art, the invention has the beneficial effects that:

(1) the method can be used for simultaneously carrying out qualitative and quantitative analysis on at least 26 bile acids with different structural types in different tissues, and has the advantages of good separation effect and higher detection result accuracy.

(2) The method can detect the bile acid content of different tissues of the animal, and the sample processing method is rapid, the method adopts methanol with certain concentration to extract and purify the bile acid in the bile, and is more beneficial to subsequent on-machine detection compared with the method adopting chloroform and the like to carry out uniform mixing and layering; when the invention is used for pretreating a plasma sample, a certain amount of triethylamine and water are added into the eluent to be compatible with methanol, so that bile acid compounds in the plasma can obtain better impurity removal and separation effects, and later detection is facilitated.

(3) In the detection method, all bile acid is well separated and responded, the quantitative limit of the bile acid can reach 2ng/ml or even lower, and a good linear relation is presented in the concentration range of 2-320 ng/ml.

Drawings

FIG. 1 is an ion flow diagram of bile acid detection of a fish plasma sample of example 4.

FIG. 2 is an ion flow diagram of HCA, beta MCA, CDCA, DCA, UDCA, HDCA, 6,7-KLCA, 7,12-KLCA, T beta MCA, T omega MCA, THDCA, TUDCA, GCA, GCDCA, GDCA, GUDCA, LCA, 7-KLCA, and 12-KLCA detection in mixed standard solutions.

FIG. 3 is an ion flow diagram of ω MCA, α MCA, and T α MCA in a mixed standard solution.

FIG. 4 is an ion flow diagram of TCA, TCDCA, CA, TDCA in a mixed standard solution.

Fig. 5 is an ion flow diagram for the test of comparative example 1.

Fig. 6 is an ion flow diagram of the test of comparative example 2.

Detailed Description

The present invention will be further described with reference to the following embodiments.

The mixed internal standard solutions in the following examples were prepared as follows:

10mg of each of the control CA-D4, CDCA-D4 and LCA-D4 was weighed and dissolved in 10ml of methanol to prepare a 1mg/ml master batch.

Respectively sucking 50 mul of CA-D4, CDCA-D4, LCA-D4 standard substance mother liquor and 50 mul of methanol, fully mixing to prepare 50 mul/ml high-concentration mixed standard solution, and gradually diluting with 50% methanol solution to prepare 5 mul/ml mixed internal standard working solution.

EXAMPLE 1 pretreatment of solid samples of whole fish

Anaesthetizing live fish in Yuanle, standing at-80 deg.C for 10min, taking out, cutting into small pieces, standing at-80 deg.C until completely freezing, freeze drying with freeze drier, and pulverizing with high-speed universal pulverizer until bile acid is extracted.

Accurately weighing 100mg of processed sample into a 2ml centrifuge tube, adding 20 mul of mixed internal standard solution with the concentration of 5 mug/ml, adding 980 mul of extracting solution which is 75% ethanol solution, then violently shaking for 3min by using a vortex oscillator to fully extract bile acid, after shaking uniformly, centrifuging for 10min (15000rpm,4 ℃) to obtain 500 mul of supernatant into a new 2ml centrifuge tube, adding 500 mul of glacial acetonitrile, centrifuging for 10min under the same conditions after uniform vortex mixing, obtaining the supernatant, passing the supernatant through a 0.2 mu m filter membrane into an automatic sample feeding bottle, and detecting by using a machine.

Example 2 pretreatment of solid samples liver, gut content and parotid gland

Preparing a clean mortar and enough liquid nitrogen, fully grinding about 200mg of frozen samples of liver, intestinal tract contents or parotid gland tissues, and waiting for extracting bile acid.

Accurately weighing 100mg of processed sample into a 2ml centrifuge tube, adding 20 mul of mixed internal standard solution with the concentration of 5 mug/ml, adding 980 mul of extracting solution which is 75% ethanol solution, then violently shaking for 3min by using a vortex oscillator to fully extract bile acid, after shaking uniformly, centrifuging for 10min (15000rpm,4 ℃) to obtain 500 mul of supernatant to a new 2ml centrifuge tube, adding 500 mul of pre-cooled glacial acetonitrile, after uniform vortex mixing, centrifuging for 10min under the same conditions, taking the supernatant to pass through a 0.2 mu m filter membrane to an automatic sample feeding bottle, and performing detection by using a machine.

EXAMPLE 3 pretreatment of liquid sample bile

Taking the gall bladder out of a refrigerator at minus 80 ℃, thawing the gall bladder on ice, taking 5 mu l of bile into a 2ml centrifuge tube, adding 1995 mu l of 80% methanol aqueous solution, uniformly vortexing, standing at minus 20 ℃ for 30min, centrifuging for 10min (15000rpm,4 ℃), taking 400 mu l of supernatant into a new 2ml centrifuge tube, adding 600 mu l of 30% methanol solution and 10 mu l of mixed internal standard solution with the concentration of 5 mu g/ml, vortexing for 1min, passing through a 0.2 mu m filter membrane to an automatic sample feeding bottle, and waiting for detection on a machine.

EXAMPLE 4 Pre-treatment of liquid sample plasma

Thawing fish plasma on ice, collecting 100 μ l plasma into 2ml centrifuge tube, adding 10 μ l 1 μ g/ml mixed internal standard solution, mixing, adding 890 μ l 75% ethanol water solution, vortexing for 1min, and storing in 4 deg.C refrigerator for use. Adopting a C18 needle type solid phase extraction filter to purify a plasma sample, wherein the plasma purification process comprises the following steps:

and (3) activation: 2ml acetonitrile, 2ml 3% formic acid water

Loading: 1ml of the above plasma sample

Leaching: 1ml of 50% methanol solution, discarding the eluate

And (3) elution: 2ml triethylamine-water-methanol (2% -18% -80%)

Mixing and collecting eluate, blowing the solvent under nitrogen flow, re-dissolving with 200 μ l 75% ethanol solution, filtering with 0.2 μm filter membrane to automatic sampling bottle, and detecting with computer.

Example 5 high performance liquid chromatography-Mass Spectrometry analysis of sample bile acids

Examples 1-4 samples were pretreated and analyzed by HPLC-MS:

an ACQUITY UPLC BEH C18 (1.7. mu. 2.1X 150mm) column was used with mobile phases A0.1% formic acid water and B0.1% formic acid acetonitrile. The gradient elution procedure is shown in table 1 below:

TABLE 1 gradient elution schedule

The flow rate was 350. mu.l/min. The column temperature was 40 ℃ and the injection volume was 3. mu.L.

Mass spectrum detection and analysis: Q-TOF detection (SCIEX, USA) was electrospray ionization (ESI) performed in negative ion mode with nebulizer pressure of 50 psi; collision energy 60V; capillary voltage 5.5 kV; the ion temperature is 600 ℃; the mass range m/z is 50-800 Da.

On the basis of this method, all bile acids are well separated and responded. The analytical chromatogram of example 4 is shown in FIG. 1.

The invention measures the lowest quantitative limit and linear range of each component according to the 2020 edition Chinese pharmacopoeia quality analysis methodology, and the results are shown in the following table 2.

The standard solutions in the linear assay were prepared as follows:

(1) 10mg of each of control TCA, TCDCA, CA, TDCA, HCA, beta MCA, alpha MCA, T alpha MCA, CDCA, DCA, UDCA, HDCA, 6,7-KLCA, 7,12-KLCA, T beta MCA, T omega MCA, THDCA, TUDCA, GCA, GCDCA, GDCA, GUDCA, LCA, 7-KLCA, 12-KLCA, CA-D4, CDCA-D4 and LCA-D4 was weighed, and 10ml of methanol was added to dissolve the reference substances to prepare a 1mg/ml master solution of the standard substance.

50 mul of HCA, omega MCA, beta MCA, alpha MCA, T alpha MCA, CDCA, DCA, UDCA, HDCA, 6,7-KLCA, 7,12-KLCA, T beta MCA, T omega MCA, THDCA, TUDCA, GCA, GCDCA, GDCA, GUDCA, LCA, 7-KLCA, 12-KLCA standard mother liquor and 50 mul of methanol are respectively sucked and fully mixed to prepare a high-concentration mixed standard solution of 50 mul/ml, and then the high-concentration mixed standard solution is gradually diluted to the standard working solution 1 of 320, 160, 80, 40, 20, 10 and 2ng/ml by using a 50 percent methanol solution. 80ng/ml of the standard working solution 1 was analyzed according to the HPLC-MS method of example 5, as shown in FIGS. 2 to 3.

Respectively sucking 50 mul of TCA, TCDCA, CA, TDCA standard substance mother liquor and 50 mul of methanol, fully mixing to prepare 50 mul/ml high-concentration mixed standard solution, and gradually diluting to 800, 400, 200, 100, 50, 25 and 10ng/ml standard working solution 2 by using 50% methanol solution. Standard working solution 1 at 200ng/ml was analyzed according to the HPLC-MS method of example 5, as shown in FIG. 4.

TABLE 2 detection, quantitation and Linear Range of different bile acids

Comparative example 1

This comparative example is compared to example 5, and the gradient elution procedure is different, as shown in table 3 below. The detection chromatogram of the mixed standard solution is shown in FIG. 5.

TABLE 3 gradient elution schedule

Comparative example 2

In comparison with example 5, the mobile phase A is 0.1% formic acid water, the mobile phase B is acetonitrile, the flow rate is 0.5ml/min, the column temperature is 55 ℃, all the reference substances have peaks in 10min, the overlapping interference exists among a plurality of chromatographic peaks, and the detected chromatographic ion flow diagram is shown in FIG. 6.

The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims

1. A liquid chromatography mass spectrometry detection method of bile acid in multiple tissues is characterized by comprising the following steps of liquid chromatography mass spectrometry analysis, wherein the mobile phase A in the liquid chromatography condition is 0.05-0.2% of formic acid water, the mobile phase B in the liquid chromatography condition is 0.05-0.2% of formic acid acetonitrile,

the gradient elution procedure was as follows:

0min，A：B，74-76：24-26，V/V；

4min，A：B，74-76：24-26，V/V；

16min，A：B，58-62：38-42，V/V；

30min，A：B，24-26：74-76，V/V；

31min，A：B，0-2：98-100，V/V；

33min，A：B，0-2：98-100，V/V；

the flow rate is 340-.

2. The liquid chromatography mass spectrometry detection method of claim 1, wherein the conditions of the mass spectrometry analysis comprise: adopting an electrospray ion source, scanning negative ions, and controlling the pressure of an atomizer to be 48-52 psi; collision energy 58-62V; the capillary voltage is 5.2-5.7 kV; the ion temperature is 590-610 ℃; the mass range m/z is 50-800 Da.

3. The method for detecting mass spectrum of liquid chromatogram according to claim 1, wherein the sample amount in the liquid chromatogram condition is 2-5 μ L, and the chromatographic column is a carbon eighteen-filler chromatographic column.

4. The liquid chromatography mass spectrometry detection method of claim 1, further comprising the preparation of a mixed internal standard working solution, specifically comprising: and adding methanol into the CA-D4, the CDCA-D4 and the LCA-D4 to prepare mixed internal standard working solution with the concentration of each component being 4-6 mu g/ml respectively.

5. The method for liquid chromatography mass spectrometry according to claim 1, further comprising preparing a standard solution, specifically comprising adding HCA, ω MCA, α MCA, T α MCA, β MCA, CDCA, DCA, UDCA, HDCA, 6,7-KLCA, 7,12-KLCA, T β MCA, T ω MCA, THDCA, TUDCA, GCA, GCDCA, GDCA, GUDCA, LCA, 7-KLCA, 12-KLCA to methanol to prepare a standard solution 1 having a concentration of each component of 2 to 320 ng/ml; adding methanol into TCA, TCDCA, CA and TDCA to prepare standard solution 2 with concentration of each component of 10-800 ng/ml.

6. The liquid chromatography-mass spectrometry detection method of claim 1, further comprising pre-treating a bile acid sample to be detected, wherein when the bile acid sample to be detected is a solid tissue, the pre-treating step comprises: adding the mixed internal standard working solution into the solid tissue, adding 70-80% ethanol solution, crushing, centrifuging, taking supernatant 1, adding glacial acetonitrile, centrifuging for the second time, and taking supernatant 2 to obtain the final product.

7. The liquid chromatography mass spectrometry detection method of claim 6, wherein the mass-to-volume ratio of the solid tissue to the 70-80% ethanol solution is 90-110mg/ml, and the crushing is performed by intense shaking uniformly by using a sample crushing system; the volume ratio of the supernatant 1 to the glacial acetonitrile is 48-52: 48-52.

8. The liquid chromatography mass spectrometry detection method of claim 6, wherein when the bile acid sample to be detected is bile, the pretreatment step comprises: adding 75-85% methanol water solution into bile, standing at-18 deg.C to-22 deg.C, centrifuging to obtain supernatant 3, adding 15-45% methanol solution and mixed internal standard working solution, centrifuging again, and collecting supernatant 4.

9. The liquid chromatography mass spectrometry detection method as claimed in claim 8, wherein the volume ratio of the bile to the 75-85% methanol aqueous solution is 1:395-405, the standing time is 25-35min, and the volume ratio of the supernatant 3 to the 15-45% methanol solution and the mixed internal standard working solution is 38-42:58-62: 1.

10. The detection method of liquid chromatography mass spectrometry according to claim 6 or 8, wherein the temperature of the centrifugation or the secondary centrifugation is 2-5 ℃, the rotation speed is 12000-18000rpm, and the time is 5-15 min.

11. The liquid chromatography mass spectrometry detection method of claim 6, wherein when the bile acid sample to be detected is a plasma sample, the pretreatment step comprises: adding the mixed internal standard solution into the plasma, mixing, adding 70-80% ethanol water solution, centrifuging, collecting supernatant, performing C18 solid phase extraction, rinsing with 45-55% methanol solution, discarding, eluting with triethylamine-water-methanol mixed solution system, and collecting eluate.

12. The method for liquid chromatography-mass spectrometry detection according to claim 10, wherein the volume ratio of the plasma to 70-80% aqueous ethanol is 1:8-10, the C18 solid phase is activated with acetonitrile and 3% aqueous formic acid at a mass ratio of 1:0.8-1.2 before extraction, and the volume ratio of triethylamine, water and methanol is 1-3:16-20: 77-83.

13. Use of a liquid chromatography mass spectrometry detection method according to any one of claims 1 to 12 for the qualitative or quantitative detection of multiple bile acids in animal tissues or chinese herbs.