CN111307993B

CN111307993B - Method for detecting bile acid content in blood

Info

Publication number: CN111307993B
Application number: CN202010313986.3A
Authority: CN
Inventors: 贾永娟; 许丽; 刘杏立; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2023-07-04
Anticipated expiration: 2040-04-20
Also published as: CN111307993A

Abstract

The invention provides a method for detecting the content of bile acid in blood, which comprises the following steps: respectively detecting mixed standard solutions with at least three concentrations under preset detection conditions by using a liquid chromatograph-mass spectrometer to obtain corresponding first detection results; wherein the mixed standard solution is a solution with 18 bile acids and an internal standard substance; fitting a standard curve equation corresponding to each bile acid according to each first detection result, an internal standard substance in the mixed standard solution and concentration of each bile acid; adding an internal standard substance into a blood sample to be tested, and carrying out high-speed centrifugation; detecting the supernatant after centrifugal treatment under the detection condition by using a liquid chromatograph-mass spectrometer to obtain a second detection result corresponding to the blood sample to be detected; and obtaining the concentration of 18 bile acids in the blood sample to be tested based on each standard curve equation and the second detection result. The scheme can detect various bile acids more rapidly and accurately.

Description

Method for detecting bile acid content in blood

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting the content of bile acid in blood.

Background

Bile acids are produced by cholesterol metabolism and can be classified into primary bile acids directly synthesized from cholesterol as a raw material and secondary bile acids produced by metabolism according to synthetic pathways. Studies have found that liver and gastrointestinal diseases can affect bile acid synthesis and processing. At present, the total concentration of the serum bile acid is mainly used as the diagnostic index of diseases such as acute and chronic hepatitis, enteritis, liver cirrhosis, neonatal jaundice, intrahepatic cholestasis in gestation period and the like in clinic. The concentration of total bile acid is detected simply, conveniently and rapidly, but the detection result has limited guiding effect on diagnosis and treatment of diseases.

With the deep research, the degree of attention of people to the metabolism, regulation and physiological function and pathological action of bile acid is improved, and how to establish a rapid and accurate determination method of various bile acids has very important research value and clinical significance.

Disclosure of Invention

The embodiment of the invention provides a method for detecting the content of bile acid in blood, which can detect various bile acids more rapidly and more accurately.

The embodiment of the invention provides a method for detecting the content of bile acid in blood, which comprises the following steps:

preparing at least three concentrations of mixed standard solutions, wherein the mixed standard solutions are solutions with 18 bile acids and internal standard substances, and the concentrations of the internal standard substances in the at least three concentrations of mixed standard solutions are the same, and the 18 bile acids comprise: lithocholic acid LCA, deoxycholic acid DCA, glycolithocholic acid GLCA, glycodeoxycholic acid GDCA, glycochenodeoxycholic acid GCDCA, glycoursodeoxycholic acid GUDCA, glycohyodeoxycholic acid GHDCA, glycocholic acid GCA, taurocholic acid TLCA, taurodeoxycholic acid TDCA, taurochenodeoxycholic acid TCDCA, taurochenodeoxycholic acid TUDCA, taurocholic acid TCA, chenodeoxycholic acid CDCA, ursodeoxycholic acid UDCA, hyodeoxycholic acid HDCA, cholic acid CA, taurochenodeoxycholic acid THDCA;

respectively detecting each mixed standard solution by using a liquid chromatograph-mass spectrometer under a preset detection condition to obtain first detection results respectively corresponding to the mixed standard solutions with at least three concentrations;

fitting standard curve equations corresponding to each bile acid respectively according to the first detection results, the internal standard substance in the mixed standard solution and the concentration of each bile acid;

adding an internal standard with the same amount as that in the mixed standard solution into a blood sample to be detected, performing high-speed centrifugation, and taking supernatant after the centrifugation as a sample to be detected;

detecting the sample to be detected under the detection condition by using a liquid chromatograph-mass spectrometer to obtain a second detection result corresponding to the blood sample to be detected;

and obtaining the concentration of the 18 bile acids in the blood sample to be tested based on each standard curve equation and the second detection result.

Preferably, the method comprises the steps of,

chromatographic conditions among the detection conditions include:

c18 reversed phase chromatographic column with column temperature of 55-60deg.C and flow rate of 0.5mL/min;

the mobile phase A is 3-7mmol/L ammonium acetate aqueous solution, and the mobile phase B is methanol: acetonitrile = 1:1 (v/v) and the mobile phase is eluted with a gradient.

Specifically, the C18 reverse phase chromatography column comprises Shim-pack Velox SP-C18 chromatography column (2.1 mm. Times.100 mm,1.8 μm) of Shimadzu corporation.

Mobile phase A aqueous ammonium acetate solution 3-7mmol/L refers to any concentration in the range of 3mmol/L to 7mmol/L, such as 3mmol/L, 3.5mmol/L, 4mmol/L, 4.8mmol/L, 5mmol/L, 5.3mmol/L, 5.5mmol/L, 5.8mmol/L, and 6mmol/L.

It is understood that the column temperature of 55-60℃means any temperature ranging from 55℃to 60℃such as 55℃56℃57℃58℃59℃and 60 ℃.

In particular, the method comprises the steps of,

mass spectrometry conditions among the detection conditions include:

adopting an atmospheric pressure chemical ionization source ESI, a positive ion scanning mode, a multi-reaction monitoring mode, wherein the temperature of a desolventizing pipe is 250-300 ℃, the temperature of a heating module is 450-500 ℃, the flow rate of atomizing gas is 2.0-3.0L/min, the flow rate of drying gas is 8-10.0L/min, the flow rate of heating gas is 8-10.0L/min, and the interface temperature is 400 ℃.

It is understood that the desolventizing pipe temperature of 250-300 c means any temperature ranging from 250 c to 300 c, such as 250 c, 260 c, 270 c, 280 c, 290 c and 300 c.

The heating module temperature of 450-500 ℃ refers to any temperature in the range of 450 ℃ to 500 ℃, such as 450 ℃, 460 ℃, 470 ℃, 480 ℃, 490 ℃ and 500 ℃.

The atomizing gas flow rate of 2.0-3.0L/min means any flow rate in the range of 2.0L/min to 3.0L/min, such as 2.0L/min, 2.2L/min, 2.4L/min, 2.6L/min, 2.8L/min and 3.0L/min.

The drying gas flow rate of 8-10.0L/min means any flow rate in the range of 8L/min to 10L/min, such as 8L/min, 8.2L/min, 8.4L/min, 8.6L/min, 8.8L/min, 9.0L/min, 9.2L/min, 9.4L/min, 9.6L/min, 9.8L/min and 10L/min.

Heating gas flow rate 8-10.0L/min refers to any flow rate in the range of 8L/min to 10L/min, such as 8L/min, 8.2L/min, 8.4L/min, 8.6L/min, 8.8L/min, 9.0L/min, 9.2L/min, 9.4L/min, 9.6L/min, 9.8L/min, and 10L/min.

Preferably, the method comprises the steps of,

the internal standard comprises: LCA-corresponding internal standard LCA-D4, DCA-corresponding internal standard DCA-D5, CDCA-corresponding internal standard CDCA-D4, UDCA-corresponding internal standard UDCA-D4, HDCA-corresponding internal standard HDCA-D5, CA-corresponding internal standard CA-D4, GLCA-corresponding internal standard CA-D4, GDCA-corresponding internal standard CA-D4, GCDCA-corresponding internal standard GCDCA-D5, GUDCA-corresponding internal standard GUDCA-D7, GHDCA-corresponding internal standard GUDCA-D7, GCA-corresponding internal standard GCA-D5, TLCA-corresponding internal standard TLCA-D5, TDCA-corresponding internal standard TCDCA-D5, TCDCA-corresponding internal standard TCDCA-D5, TUDCA-corresponding internal standard TUDCA-D5, THDCA-corresponding internal standard TUDCA-D5, and TCA-corresponding internal standard TCA-D5.

Specifically, the solutions of 13 kinds of internal standards corresponding to 18 kinds of bile acids, namely LCA-D4, DCA-D5, CDCA-D4, UDCA-D4, HDCA-D5, CA-D4, GCDCA-D5, GUDCA-D7, GCA-D5, TLCA-D5, TCDCA-D5, TCA-D5 and TUDCA-D5, can be used for various kinds of bile acids to simplify the test method of bile acids, for example, GLCA and GDCA share the same internal standard CA-D4, TDCA and TCDCA share the same internal standard TCDCA-D5, TUDCA and THDCA share the same internal standard TCDCA-D5, and other bile acids can be used as internal standards, for example, the internal standard of THDCA is the isotope of THDCA.

Preferably, the method comprises the steps of,

the two variables of the standard curve equation corresponding to each bile acid are respectively: the ratio of the chromatographic peak area of the bile acid to the chromatographic peak area of the internal standard corresponding to the bile acid, and the ratio of the concentration of the bile acid to the concentration of the internal standard corresponding to the bile acid.

Specifically, two variables of the standard curve equation corresponding to LCA are respectively: a ratio of a chromatographic peak area of the LCA to a chromatographic peak area of an internal standard corresponding to the LCA, and a ratio of a concentration of the LCA to a concentration of the internal standard corresponding to the LCA;

the two variables of the standard curve equation corresponding to DCA are respectively: the ratio of the chromatographic peak area of DCA to the chromatographic peak area of the internal standard corresponding to DCA, and the ratio of the concentration of DCA to the concentration of the internal standard corresponding to DCA;

the two variables of the standard curve equation corresponding to GLCA are respectively: a ratio of a chromatographic peak area of GLCA to a chromatographic peak area of an internal standard corresponding to GLCA, and a ratio of a concentration of GLCA to a concentration of an internal standard corresponding to GLCA;

the two variables of the standard curve equation corresponding to GDCA are respectively: the ratio of the chromatographic peak area of GDCA to the chromatographic peak area of the internal standard corresponding to GDCA, and the ratio of the concentration of GDCA to the concentration of the internal standard corresponding to GDCA;

the two variables of the standard curve equation corresponding to GCDCA are respectively: the ratio of the chromatographic peak area of GCDCA to the chromatographic peak area of the internal standard corresponding to GCDCA, and the ratio of the concentration of GCDCA to the concentration of the internal standard corresponding to GCDCA;

the two variables of the standard curve equation corresponding to GUDCA are respectively: the ratio of the chromatographic peak area of GUDCA to the chromatographic peak area of the internal standard corresponding to GUDCA, and the ratio of the concentration of GUDCA to the concentration of the internal standard corresponding to GUDCA;

the two variables of the standard curve equation corresponding to GHDCA are respectively: the ratio of the chromatographic peak area of GHDCA to the chromatographic peak area of the internal standard corresponding to GHDCA, and the ratio of the concentration of GHDCA to the concentration of the internal standard corresponding to GHDCA;

the two variables of the standard curve equation corresponding to the GCA are respectively: a ratio of a chromatographic peak area of the GCA to a chromatographic peak area of an internal standard corresponding to the GCA, and a ratio of a concentration of the GCA to a concentration of the internal standard corresponding to the GCA;

the two variables of the standard curve equation corresponding to TLCA are respectively: a ratio of a chromatographic peak area of TLCA to a chromatographic peak area of an internal standard corresponding to TLCA, and a ratio of a concentration of TLCA to a concentration of an internal standard corresponding to TLCA;

the two variables of the standard curve equation corresponding to TDCA are respectively: a ratio of a chromatographic peak area of TDCA to a chromatographic peak area of an internal standard corresponding to TDCA, and a ratio of a concentration of TDCA to a concentration of an internal standard corresponding to TDCA;

the two variables of the standard curve equation corresponding to TCDCA are respectively: the ratio of the chromatographic peak area of TCDCA to the chromatographic peak area of the internal standard corresponding to TCDCA, and the ratio of the concentration of TCDCA to the concentration of the internal standard corresponding to TCDCA;

the two variables of the standard curve equation corresponding to TUDCA are: the ratio of the chromatographic peak area of TUDCA to the chromatographic peak area of the internal standard corresponding to TUDCA, and the ratio of the concentration of TUDCA to the concentration of the internal standard corresponding to TUDCA;

the two variables of the standard curve equation corresponding to TCA are respectively: a ratio of a chromatographic peak area of TCA to a chromatographic peak area of an internal standard corresponding to TCA, and a ratio of a concentration of TCA to a concentration of an internal standard corresponding to TCA;

the two variables of the standard curve equation corresponding to CDCA are respectively: a ratio of a chromatographic peak area of CDCA to a chromatographic peak area of an internal standard corresponding to CDCA, and a ratio of a concentration of CDCA to a concentration of an internal standard corresponding to CDCA;

the two variables of the standard curve equation corresponding to UDCA are respectively: a ratio of a chromatographic peak area of UDCA to a chromatographic peak area of an internal standard corresponding to UDCA, and a ratio of a concentration of UDCA to a concentration of an internal standard corresponding to UDCA;

the two variables of the standard curve equation corresponding to HDCA are respectively: a ratio of a chromatographic peak area of HDCA to a chromatographic peak area of an internal standard corresponding to HDCA, and a ratio of a concentration of HDCA to a concentration of an internal standard corresponding to HDCA;

the two variables of the standard curve equation corresponding to CA are respectively: a ratio of a chromatographic peak area of CA to a chromatographic peak area of an internal standard corresponding to CA, and a ratio of a concentration of CA to a concentration of an internal standard corresponding to CA;

the two variables of the standard curve equation corresponding to THDCA are respectively: the ratio of the chromatographic peak area of THDCA to the chromatographic peak area of the internal standard corresponding to THDCA, and the ratio of the concentration of THDCA to the concentration of the internal standard corresponding to THDCA.

Preferably, the method comprises the steps of,

the gradient elution includes:

in this embodiment, the gradient elution is adopted to better separate 18 kinds of bile acids in the blood sample to be measured, namely corresponding internal standard substances, so as to determine the respective corresponding concentrations of the 18 kinds of bile acids in the blood sample to be measured based on the chromatographic peak areas of the bile acids and the corresponding internal standard substances and the concentrations of the internal standard substances.

Preferably, the method comprises the steps of,

the preparation of the mixed standard solution with at least three concentrations comprises the following steps:

respectively weighing 18 kinds of bile acid standard substances, and respectively dissolving the 18 kinds of bile acid standard substances by using methanol to obtain target object standard stock solutions corresponding to each kind of bile acid;

mixing the standard stock solutions of the 18 bile acids according to different proportions, and respectively diluting by using a diluent to obtain at least three standard yeast mixed working solutions with different concentrations;

respectively weighing standard substances of the internal standard substances corresponding to the 18 bile acids, and dissolving the standard substances by using methanol to obtain an internal standard stock solution of the internal standard substances;

transferring the internal standard stock solution, and diluting the transferred internal standard stock solution by using the diluent to obtain an internal standard working solution;

and respectively transferring each standard yeast mixed working solution with the same volume, adding the same amount of the internal standard working solution and the diluent into each standard yeast mixed working solution, respectively and uniformly vortex and mix for 1-2min at the rotating speed of 1500-2000rpm, and taking the supernatant after vortex as a mixed standard solution.

Specifically, when 18 kinds of bile acid standard substances are dissolved respectively, the volume ratio of the available bile acid standard substance to the bile acid standard substance is 0.7:1-1:1, so that a small amount of solvent is fully used for dissolving the standard substance of the bile acid to the greatest extent, and the methanol solvent is used as a conventional solvent, and the methanol solvent is used as a dissolving agent, so that the dissolving operation of the bile acid can be simplified. Likewise, for the standard of the internal standard, a volume ratio of 0.7 to the standard of the internal standard is also available: 1-1:1 in order to simplify the dissolution operation of the standard of the internal standard. In order to facilitate calculation of concentration of bile acid in a blood sample to be measured, the same amount of internal standard working solution and diluent can be added into standard mixed working solutions with different concentrations to obtain a solution meeting the test concentration of a liquid chromatography-mass spectrometer, then vortex treatment is carried out, and supernatant is used as a mixed standard solution to remove trace impurities in the internal standard working solution, the standard mixed working solution and the diluent, so that the accuracy of the test is prevented from being influenced.

When the internal standard substance is plural, methanol may be used to dissolve the standard substance of the internal standard substance.

Specifically, the prepared standard stock solution of the target object and the standard yeast mixed working solution can be stored at the temperature of between 80 ℃ below zero and 20 ℃ below zero, and the internal standard stock solution and the internal standard working solution can be stored at the temperature of between 80 ℃ below zero and 4 ℃ below zero.

Preferably, the method comprises the steps of,

the diluent comprises: 25% of methanol-acetonitrile and 75% of 3-7mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the methanol-acetonitrile is 1:1.

specifically, the initial eluting concentrations of the a phase and the B phase of the eluting solvent can be used as the diluents of the standard stock solution of the dilution target object and the standard stock solution of the internal standard to reduce the interference of other reagents.

Preferably, the method comprises the steps of,

after the internal standard substance with the same amount as that in the mixed standard solution is added into the blood sample to be detected, before the high-speed centrifugation is carried out and the supernatant after the centrifugation is taken as the sample to be detected, the method further comprises the steps of:

adding a precipitated protein reagent into the blood sample to be tested;

the high-speed centrifugal treatment is carried out, supernatant fluid after the centrifugal treatment is taken as a sample to be detected, and the method comprises the following steps:

vortex mixing at 1500-2000rpm for 2-5min, centrifuging at 10000-15000rpm for 4-6min, and removing the first supernatant;

blowing the first supernatant with nitrogen, adding a preset amount of complex solution, vortex mixing for 1-3min at a rotating speed of 1500-2000rpm, centrifuging for 4-6min at a high speed of 10000-15000rpm, and taking the centrifuged second supernatant as a sample to be detected.

Specifically, in order to avoid that components such as fibrinogen in blood of a user to be tested affect the accuracy of bile acid test, a blood sample collected from a human body may be treated, for example, at least 2mL of blood to be tested is taken first, and centrifuged at 3500rpm for 10min, and a supernatant is taken as the blood sample to be tested. And then adding a protein precipitation reagent to precipitate protein in the blood sample to be tested. Because reagents such as a precipitated protein reagent and an internal standard substance solution are added into the blood sample to be tested, the concentration of the blood sample to be tested is thinner at the moment, in order to more accurately test the concentration of bile acid in the blood sample to be tested, the blood sample to be tested can be concentrated in a nitrogen blow-drying mode, then the re-dissolved solution is utilized to re-dissolve the blood sample to be tested to obtain the concentration reaching the test requirement, and vortex is carried out to uniformly mix the blood sample to be tested with the re-dissolved solution.

Preferably, the method comprises the steps of,

the complex solution comprises: 25% of methanol-acetonitrile and 75% of 3-7mmol/L mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the methanol-acetonitrile is 1:1.

specifically, the initial elution concentrations of the A phase and the B phase of the eluting solvent are used as a re-solution of the blood sample to be tested after re-dissolving and drying so as to reduce the interference of other reagents.

Preferably, the method comprises the steps of,

the precipitated protein reagent comprises: acetonitrile, wherein, acetonitrile and the volume ratio of blood sample that awaits measuring is 3:1-10:1, so as to better precipitate protein in blood to be detected, and reduce detection of bile acid in blood samples to be detected by other substances.

The invention provides a method for detecting the content of bile acid in blood, which combines an internal standard method with a high performance liquid chromatography-tandem mass spectrometry, wherein the method uses protein precipitation for pretreatment, the pretreatment method is simple, the sample treatment time is shortened, and meanwhile, the advantage of multi-reaction monitoring by using a mass spectrometry technology is utilized, so that interference factors are greatly reduced, the specificity is strong, the sensitivity is high, the detection result is more accurate, and meanwhile, the analysis time is short.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for detecting bile acid content in blood according to an embodiment of the present invention;

FIG. 2 is a chart of bile acid in a mixed standard solution according to an embodiment of the present invention;

FIG. 3 is a graph of an internal standard in a mixed standard solution according to an embodiment of the present invention;

FIG. 4 is a chart of bile acid in a sample to be tested according to an embodiment of the present invention;

fig. 5 is a diagram of an internal standard in a sample to be tested according to an embodiment of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.

As shown in fig. 1, an embodiment of the present invention provides a method for detecting bile acid content in blood, including:

step 101: preparing at least three concentrations of mixed standard solutions, wherein the mixed standard solutions are solutions with 18 bile acids and internal standard substances, and the concentrations of the internal standard substances in the at least three concentrations of mixed standard solutions are the same, and the 18 bile acids comprise: lithocholic acid LCA, deoxycholic acid DCA, glycolithocholic acid GLCA, glycodeoxycholic acid GDCA, glycochenodeoxycholic acid GCDCA, glycoursodeoxycholic acid GUDCA, glycohyodeoxycholic acid GHDCA, glycocholic acid GCA, taurocholic acid TLCA, taurodeoxycholic acid TDCA, taurochenodeoxycholic acid TCDCA, taurochenodeoxycholic acid TUDCA, taurocholic acid TCA, chenodeoxycholic acid CDCA, ursodeoxycholic acid UDCA, hyodeoxycholic acid HDCA, cholic acid CA, taurochenodeoxycholic acid THDCA;

step 102: respectively detecting each mixed standard solution by using a liquid chromatograph-mass spectrometer under a preset detection condition to obtain first detection results respectively corresponding to the mixed standard solutions with at least three concentrations;

step 103: fitting standard curve equations corresponding to each bile acid respectively according to the first detection results, the internal standard substance in the mixed standard solution and the concentration of each bile acid;

step 104: adding an internal standard with the same amount as that in the mixed standard solution into a blood sample to be detected, performing high-speed centrifugation, and taking supernatant after the centrifugation as a sample to be detected;

step 105: detecting the sample to be detected under the detection condition by using a liquid chromatograph-mass spectrometer to obtain a second detection result corresponding to the blood sample to be detected;

step 106: and obtaining the concentration of the 18 bile acids in the blood sample to be tested based on each standard curve equation and the second detection result.

The detection process of the liquid chromatography-mass spectrometer used in the detection method provided by the embodiment of the invention can be that a standard curve equation between the chromatographic peak area of the standard target object and the concentration of the standard target object is constructed based on the relation between the chromatographic peak area of the standard target object and the concentration of the standard target object in the mixed standard solution, and then the chromatographic peak area of the target object in the blood sample to be detected is substituted into the standard curve equation, so that the concentration of the target object in the blood sample to be detected can be calculated.

However, due to the processing process and the detection error of the blood sample to be measured, a standard curve equation constructed based on the chromatographic peak area of the standard target object and the concentration of the standard target object has a certain deviation, and then the concentration of the target object in the blood sample to be measured calculated based on the standard curve equation also has a deviation. More preferably, in the detection process of the liquid chromatograph-mass spectrometer used in the detection method, the ratio of the chromatographic peak area of the target object to the chromatographic peak area of the internal standard object is used as a first independent variable, the ratio of the concentration of the target object to the concentration of the internal standard object is used as a second independent variable, a linear regression fit standard curve equation is performed through at least three groups (the first independent variable and the second independent variable), and then the ratio of the chromatographic peak area of the target object (namely the 18 bile acids) in the blood sample to be detected to the chromatographic peak area of the internal standard object is substituted into the standard curve equation, so that the chromatographic peak area of the target object of the blood sample to be detected in the blood sample to be detected and the concentration of the internal standard object can be obtained, and the ratio of the concentration of the target object in the blood sample to be detected to the concentration of the internal standard object is calculated.

Preferably, the standard target and the target in the blood sample to be tested are LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA, THDCA.

The method for detecting 18 bile acids in human blood will be described in detail in several examples.

Example 1: preparation of mixed standard solution

Firstly, respectively weighing 18 kinds of bile acid standard substances, and respectively dissolving the 18 kinds of bile acid standard substances by using methanol to obtain target object standard stock solutions corresponding to each kind of bile acid;

mixing standard stock solutions of 18 bile acids according to different proportions, and respectively diluting with diluent (25% methanol-acetonitrile: 75% 5mM ammonium acetate aqueous solution) to obtain seven standard yeast mixed working solutions with different concentrations, and preserving at-20deg.C, wherein each standard yeast mixed working solution contains standard stock solutions of 18 bile acids.

And respectively weighing the standard substances of the 13 internal standards, dissolving the standard substances by using methanol to obtain an internal standard stock solution of the internal standard substances, and storing the internal standard stock solution at the temperature of minus 4 ℃, wherein the standard substances of the internal standard substances are LCA-D4, DCA-D5, CDCA-D4, UDCA-D4, HDCA-D5, CA-D4, GCDCA-D5, GUDCA-D7, GCA-D5, TLCA-D5, TCDCA-D5, TCA-D5 and TUDCA-D5 respectively.

Each standard stock solution was removed separately in preset amounts and mixed with 25% methanol-acetonitrile: the solution is diluted with 75% of 5mM ammonium acetate aqueous solution to obtain 2 mug/mL internal standard working solution, and the internal standard working solution is preserved at 4 ℃ for standby.

For each of the seven different concentration standard curve mixed working solutions, transferring 10 μl of the standard curve mixed working solution and 10 μl of the internal standard working solution into a centrifuge tube respectively by a pipette, and transferring 80 μl of 25% methanol-acetonitrile: diluting with 75% 5mM ammonium acetate aqueous solution, vortex mixing at 1500-2000rpm for 1-2min, and taking supernatant as mixed standard solution.

The mixed standard solutions of 18 bile acids are shown in the following table 1, and the standard curve concentration information is shown in the following table 2:

TABLE 1

Table 2 (ng/mL)

And detecting the seven mixed standard solutions with different concentrations by using a high performance liquid tandem mass spectrometer to obtain chromatograms of the standard solutions of LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA, THDCA and internal standard substances with seven different concentrations.

The chromatographic peak areas of LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA and THDCA and the corresponding internal standard were obtained from the chromatograms of the standard solutions at each concentration, respectively.

The ratio of the chromatographic peak areas of the seven different concentrations of LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA and THDCA to the chromatographic peak areas of the corresponding internal standard was taken as the ordinate yi of the standard curve equation.

The ratio of the concentrations of LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA and THDCA at the seven different concentrations to the concentrations of the corresponding internal standard is the abscissa xi (i=1, 2,3, … … 18) as a standard curve equation.

And carrying out linear regression on the seven data with different concentrations obtained by detection, and fitting to obtain a standard curve equation corresponding to LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA and THDCA respectively, wherein yi=ai, xi+bi (i=1, 2,3, … … 18), and coefficients ai and bi (i=1, 2,3, … … 18) are obtained, wherein ai is the slope of the standard curve equation corresponding to yi, and bi is the intercept of the standard curve equation corresponding to yi.

Example 2: pretreatment of collected blood

Removing at least 2ml of continuous page to be detected, centrifuging at 3500rpm for 10min, taking supernatant as blood sample to be detected, and freezing the blood sample to be detected at-80-20deg.C until analysis.

Example 3: treatment of blood samples to be tested

Transferring 10 mu L of the internal standard working solution in the embodiment 1 into a 2mL centrifuge tube by using a pipetting gun, adding 100-300 mu L of the to-be-detected blood sample in the embodiment 2, adding 3-10 times of the volume of the to-be-detected blood sample, carrying out vortex mixing for 2-5min at a rotating speed of 1500-2000rpm, carrying out high-speed centrifugation for 4-6min at a rotating speed of 10000-15000rpm, transferring a certain amount of supernatant fluid after centrifugation into a clean 1.5mL centrifuge tube, transferring the 1.5mL centrifuge tube containing the supernatant fluid into a nitrogen blow-drying device, blow-drying the supernatant fluid, transferring a certain amount of complex solution into the 1.5mL centrifuge tube with the upper clear drying, carrying out vortex mixing for 1-3min at a rotating speed of 1500-2000rpm, carrying out high-speed centrifugation for 4-6min at a rotating speed of 10000-15000rpm, and transferring 90 mu L of the supernatant fluid after centrifugation to obtain the to-be-detected sample.

Example 4: treatment of blood samples to be tested

Detecting the sample to be detected in the embodiment 3 by using a high performance liquid tandem mass spectrometer to obtain chromatograms of 18 bile acids and internal standards of the sample to be detected, obtaining 18 bile acid chromatographic peak areas and internal standard chromatographic peak areas from the 18 bile acids and internal standard chromatograms, substituting the ratio yi of the 18 bile acid chromatographic peak areas and the corresponding internal standard chromatographic peak areas into a corresponding standard curve equation yi=ai+bi (i=1, 2,3, … … 18) in the embodiment 1, and obtaining the relative concentration xi (i=1, 2,3, … …) of the 18 bile acids and the corresponding internal standards in the sample to be detected by calculating, wherein the concentration of the internal standards in the internal standard working fluid is known, thereby obtaining the 18 bile acid concentrations of the sample to be detected.

Example 5: detection conditions

The liquid phase conditions in example 1 and example 4 were:

chromatographic column: shimadzu corporation's Shim-pack Velox SP-C18 column (2.1 mm. Times.100 mm,1.8 μm);

elution conditions: mobile phase A is 5mmol/L ammonium acetate aqueous solution; phase B-methanol: acetonitrile = 1:1 (v/v), gradient elution;

analysis time: 22-25min; column temperature: 55-60 ℃; sample injection amount: 10-20 mu L; flow rate: 0.5mL/min.

The gradient elution procedure is shown in table 3 below:

the tandem mass spectrometry conditions in example 1 and example 4 were:

atmospheric pressure chemical ionization source ESI, positive ion scan mode, multiple reaction monitoring mode (MRM), desolventizing tube temperature: 300 ℃; heating module temperature: 500 ℃; atomizing gas flow rate: 3.0L/min; drying gas flow rate: 10.0L/min; heating air flow rate: 10.0L/min; interface temperature: 400 ℃;

the parameters of the mass spectrometer in the liquid chromatograph-mass spectrometer are shown in the following table 4:

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example 6: linear relation and quantitative limit of the above detection method

The mixed standard solution of 18 bile acids was removed, 10 μl of the internal standard working solution was added, and the measurement was carried out according to the treatment and measurement conditions of this example, and the quantitative ion chromatographic peak area-concentration was plotted to obtain a standard curve, and the results show that the linear range and quantitative limit of 18 bile acids are shown in the following table 5:

table 5:

example 7: recovery rate and precision of the above detection method

And (3) adding mixed standard solutions with low, medium and high concentration levels into the sample to be detected, measuring according to the method of the embodiment, repeating the blank control sample and the standard adding sample with each concentration level for 3 batches, carrying out sample injection analysis, and calculating the standard adding recovery rate and precision of the method. The results are shown in tables 6 to 8 below, and the results show that the recovery rate of the remaining bile acids is good, except LCA, and RSD <4.5% is within 80% -120%, with good precision.

TABLE 6

TABLE 7

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TABLE 8

In combination with the verification test, the recovery rate, the detection limit, the precision and other technical indexes of the embodiment meet the requirements, and the method simultaneously detects 18 bile acids in human serum/plasma, has good repeatability and good sample addition recovery rate, thereby improving the accuracy of the detection result and eliminating the systematic error.

Fig. 2 is a graph of 18 bile acids LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA, THDCA in a mixed standard solution.

Wherein, the retention time of the 18 bile acids is shown in table 9:

TABLE 9

Compounds of formula (I)	Retention time (min)
		LCA	17.647
DCA	15.582
		CDCA	15.307
UDCA	11.333
		HDCA	12.27
CA	11.972
		GLCA	14.465
GDCA	12.067
		GCDCA	11.63
GUDCA	7.934
		GHDCA	8.822
GCA	9.307
		TLCA	14.196
TDCA	12.074
		TCDCA	11.658
TUDCA	8.191
		THDCA	8.931
TCA	9.468

FIG. 3 is a graph of internal standards LCA-D4, DCA-D5, CDCA-D4, UDCA-D4, HDCA-D5, CA-D4, GCDCA-D5, GUDCA-D7, GCA-D5, TLCA-D5, TCDCA-D5, TCA-D5, TUDCA-D5 in a mixed standard solution.

Wherein, the retention time of the 13 internal standards is shown in table 10:

table 10

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FIG. 4 is a graph of 18 bile acids LCA, DCA, GLCA, GDCA, GCDCA, GUDCA, GHDCA, GCA, TLCA, TDCA, TCDCA, TUDCA, TCA, CDCA, UDCA, HDCA, CA, THDCA in a sample to be tested;

wherein, the retention time of the 18 bile acids is shown in table 11:

TABLE 11

FIG. 5 is a graph of internal standards LCA-D4, DCA-D5, CDCA-D4, UDCA-D4, HDCA-D5, CA-D4, GCDCA-D5, GUDCA-D7, GCA-D5, TLCA-D5, TCDCA-D5, TCA-D5, TUDCA-D5 in a sample to be tested.

Wherein, the retention time of the 13 internal standards is shown in table 12:

table 12

Compounds of formula (I)	Retention time (min)
		LCA-D4	17.636
DCA-D5	15.54
		CDCA-D4	15.287
UDCA-D4	11.306
		HDCA-D5	12.233
CA-D4	11.959
		GCDCA-D5	11.599
GUDCA-D7	7.883
		GCA-D5	9.274
TLCA-D5	14.161
		TCDCA-D5	11.624
TUDCA-D5	8.16
		TCA-D5	9.432

Specifically, the abscissa in fig. 2 to 5 is the acquisition time, and the ordinate is the ion signal intensity.

In this embodiment, as can be seen from fig. 2 and fig. 4, the retention time of 18 bile acids in the sample to be detected is consistent with the retention time of the corresponding bile acids in the mixed standard solution, and the method uses the isotope marker as an internal standard substance, so that the identification of the target compound is more accurate, the analysis time is short, the interference is small, the internal standard quantity is suitable for high specificity, and the accuracy and sensitivity are high. Likewise, as can be seen from fig. 3 and 5, the retention times of 13 internal standards in the sample to be tested are consistent with the retention times of corresponding internal standards in the turbidity shocking and standard solutions.

It is noted that relational terms such as first and second, and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the statement "comprises/comprising" does not exclude the presence of other like elements in a process, method, article or apparatus that comprises the element.

Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A method for detecting bile acid content in blood, comprising:

respectively transferring each standard yeast mixed working solution with the same volume, adding the same amount of the internal standard working solution and the diluent into each standard yeast mixed working solution, respectively and uniformly vortex and mix for 1-2min at the rotating speed of 1500-2000rpm, and taking the supernatant after vortex as a mixed standard solution;

the diluent comprises: 25% of methanol-acetonitrile and 75% of 3-7mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the methanol-acetonitrile is 1:1, a step of;

adding a precipitated protein reagent and an internal standard substance with the same amount as that in the mixed standard solution into a blood sample to be detected, wherein the precipitated protein reagent is acetonitrile; the volume ratio of acetonitrile to the blood sample to be measured is 3:1-10:1;

blowing the first supernatant with nitrogen, adding a preset amount of complex solution, vortex mixing for 1-3min at a rotating speed of 1500-2000rpm, centrifuging for 4-6min at a high speed of 10000-15000rpm, and taking the centrifuged second supernatant as a sample to be detected; the complex solution is as follows: 25% of methanol-acetonitrile and 75% of 3-7mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the methanol-acetonitrile is 1:1, a step of;

obtaining the concentration of the 18 bile acids in the blood sample to be tested based on each standard curve equation and the second detection result;

chromatographic conditions among the detection conditions include:

the mobile phase A is 3-7mmol/L ammonium acetate aqueous solution, and the mobile phase B is methanol: acetonitrile = 1:1 (v/v) the mobile phase is eluted with a gradient;

the gradient elution includes:

mass spectrometry conditions among the detection conditions include:

2. The method for detecting the concentration of bile acids in blood according to claim 1,

3. The method for detecting the concentration of bile acids in blood according to claim 1,