CN116183780A - Absolute quantitative analysis method for bile acid in serum sample - Google Patents
Absolute quantitative analysis method for bile acid in serum sample Download PDFInfo
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- CN116183780A CN116183780A CN202310450174.7A CN202310450174A CN116183780A CN 116183780 A CN116183780 A CN 116183780A CN 202310450174 A CN202310450174 A CN 202310450174A CN 116183780 A CN116183780 A CN 116183780A
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- acid
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- bile
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- DKPMWHFRUGMUKF-CRKPLTDNSA-N beta-muricholic acid Chemical compound C([C@H]1[C@H](O)[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DKPMWHFRUGMUKF-CRKPLTDNSA-N 0.000 description 1
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- GHCZAUBVMUEKKP-XROMFQGDSA-N glycoursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)CC1 GHCZAUBVMUEKKP-XROMFQGDSA-N 0.000 description 1
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- 238000002545 neutral loss scan Methods 0.000 description 1
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- 238000002542 parent ion scan Methods 0.000 description 1
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- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
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Abstract
The embodiment of the invention discloses an absolute quantitative analysis method of bile acid in a serum sample, which comprises the following steps: preparing a working reagent which is a solution containing isotope labels of cholic acid-D4, lithocholic acid-D4 and ursodeoxycholic acid-2, 4-D4; preparing a sample to be tested by using the prepared working reagent and a serum sample; configuring at least two calibration reagents; the calibration reagent is a solution containing thirty-eight medium bile acid as a solute; the concentration of each solute in the calibration reagent is the same, and the concentrations of at least two calibration reagents are different; determining a calibration reagent by utilizing high performance liquid chromatography-tandem mass spectrometry, and determining a fitting curve of each solute; and determining the absolute content of bile acid in the sample to be detected by utilizing high performance liquid chromatography-tandem mass spectrometry according to the determination result and the obtained fitting curve.
Description
Technical Field
The invention belongs to the technical field of compound quantitative analysis, and particularly relates to an absolute quantitative analysis method of bile acid in a serum sample.
Background
Bile acid is a generic name of cholanic acid, exists in the form of sodium salt or potassium salt, is synthesized from cholesterol in liver cells, and is a main removal pathway of steroid substances in the body. The body is in steady state equilibrium by a series of regulatory mechanisms. Bile acids can be used as signal molecules and play an important role in regulating various metabolic processes of the body. The bile acid in the biological tissue sample can be measured by various detection techniques to construct a corresponding bile acid metabolism profile, and the method has certain application in liver and gall diseases, gastrointestinal diseases, metabolic diseases, nervous system diseases and other diseases.
Blood is composed of cells including erythrocytes, leukocytes and platelets, and body fluid, which is plasma (serum) containing various proteins with specific functions and numerous small molecule metabolites. Blood circulates and reciprocates in the body, and interdependencies and influences each tissue of the human body, so that metabolic changes of physiological and pathological in the body can be reflected, and important clues or experimental basis can be provided by detecting and analyzing the metabolic products and knowing subtle differences, characteristics and accompanying phenomena.
The main body of the cerebrospinal fluid is an ultrafiltrate of blood plasma, and the blood brain barrier is different from the blood plasma, is closely related to cerebral nerve tissue, and can reflect the physiological and pathological metabolic changes of the cerebral nerve tissue.
Bile acids belong to the generic term for cholanic acids, which have both hydrophilic and hydrophobic groups in their molecules, and their alkali metal salts are readily soluble in water and alcohols. In mammals, forty-two kinds of bile acids and bile salts are commonly known because of their similar solubility and the same molecular groups, and because of their easy affinity with other substances, it is difficult to perform efficient, reliable and highly specific quantitative analysis of forty-two kinds of bile acids using conventional chromatography-mass spectrometry systems.
Disclosure of Invention
In view of this, some embodiments disclose a method for absolute quantitative analysis of bile acids in a serum sample, the method comprising:
preparing a working reagent which is a solution containing isotope labels of cholic acid-D4, lithocholic acid-D4 and ursodeoxycholic acid-2, 4-D4;
preparing a sample to be tested by using the prepared working reagent and a serum sample;
configuring at least two calibration reagents; the targeting agent is a solution comprising cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, tauchenodeoxycholic acid, deoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, lithocholic acid, glucolithocholic acid, taurocholate, hyocholic acid, gan Qing cholic acid, taurocholate, alpha-murine cholic acid, taurine-alpha-polyphenol sodium salt, beta-murine cholic acid, omega-murine cholic acid, tauro-omega-murine cholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, sodium taurochenodeoxycholic acid, 5 beta-cholic acid-3 alpha, 6 beta-diol, dehydrocholic acid, glycodehydrocholic acid, taurodeoxycholic acid, 7-keto-3 alpha, 12-alpha-dihydroxycholic acid, isodeoxycholic acid, orthocholic acid, 5-beta-cholic acid-3-alpha-alcohol-6-one, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, 12-ketolithocholic acid,12 ketochenodeoxycholic acid, nordeoxycholic acid, and isolithocholic acid as a solute; the concentration of each solute in the calibration reagent is the same, and the concentrations of at least two calibration reagents are different;
determining a calibration reagent by utilizing high performance liquid chromatography-tandem mass spectrometry, and determining a fitting curve of each solute;
and determining the absolute content of bile acid in the sample to be detected by utilizing high performance liquid chromatography-tandem mass spectrometry according to the determination result and the obtained fitting curve.
Further, some embodiments disclose an absolute quantitative method for analyzing bile acid in a serum sample, wherein the solvent of the working reagent is methanol.
In some embodiments, the absolute quantitative analysis method of bile acid in serum sample is disclosed, and the solvent of the sample to be tested is acetonitrile or isopropanol.
Some embodiments disclose an absolute quantitative analysis method of bile acid in serum sample, wherein the solvent of the calibration reagent is methanol.
The method for absolute quantitative analysis of bile acid in serum samples disclosed in some embodiments comprises the following steps:
uniformly mixing a working reagent, a serum sample and a solvent according to a set proportion;
after mixing evenly, centrifugal precipitation is carried out, supernatant fluid is taken, and nitrogen purging is utilized for drying;
and re-dissolving the sample by using a re-dissolving solvent, and taking the supernatant after re-dissolving to obtain a sample to be tested.
The absolute quantitative analysis method of bile acid in serum samples disclosed in some embodiments comprises the steps that a redissolution solvent is an acetonitrile aqueous solution of formic acid with the mass content of 0.1% and/or a methanol aqueous solution with the mass content of 60%, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1.
in some embodiments, the volume ratio of the working reagent, the serum sample and the solvent in the sample to be measured is 3:47:200.
The absolute quantitative analysis method of bile acid in serum sample disclosed in some embodiments, the test conditions of high performance liquid chromatography-tandem mass spectrometry include:
conditions of high performance liquid chromatography:
mobile phase a:0.1% formic acid in water, mobile phase B:0.1% formic acid in acetonitrile;
the separation conditions are shown in Table 1;
TABLE 1 list of conditions for high performance liquid chromatography
The tandem mass spectrum is formed by connecting three four-level rod mass spectrometers in series, and the detection conditions are as follows:
ionization mode: electrospray ionization, ESI (-;
ion spray voltage: 2.7kV;
sheath gas (Arb): 50;
auxiliary device (Arb): 10;
purge gas (Arb): 1, a step of;
ion transport capillary temperature: 320 ℃;
sprayer temperature: 150 ℃;
the detection mode is as follows: selecting reaction monitoring;
cycle time: 1.0sec;
q1 resolution: 0.7;
q3 resolution: 0.7;
collision gas: 1.5mTorr;
in-source cleavage voltage: 0V;
chromatographic peak width: 12sec.
Some embodiments disclose methods for absolute quantitative analysis of bile acids in serum samples, the determination of the fitted curve comprising:
according to the detection result of the calibration reagent, counting the area of each solute mass spectrum peak in the calibration reagent;
carrying out normalization treatment according to the base peaks to obtain the ionic strength of each solute;
and linearly fitting the ionic strength of each solute with the corresponding ionic concentration to obtain a fitted curve model.
The absolute quantitative analysis method for bile acid in serum samples disclosed in some embodiments, using the test result and the fitted curve of the sample to be tested, comprises:
according to the mass spectrum detection result of the sample to be detected, counting the mass spectrum peak area of each bile acid component in the sample to be detected;
carrying out normalization treatment according to the basic peak to obtain the ionic strength of each bile acid component;
and respectively bringing the ionic strength of each bile acid component into a fitting curve to obtain the corresponding molecular number.
According to the absolute quantitative analysis method for bile acid in the serum sample disclosed by the embodiment of the invention, the isotope label containing the object to be detected is used as an internal standard in the working reagent, so that the stability and repeatability of an analysis result are ensured, different components of the bile acid are effectively separated through the designed mobile phase chromatographic conditions, the mutual interference among different components is reduced, the interference of a solvent and a mobile phase is reduced, and the reliability of specificity and quantitative detection is ensured; the sensitivity and the specificity of the analysis are ensured through the tandem mass spectrum, the serum sample with low sample quantity is utilized by using the calibration reagent with different components and concentrations, the absolute quantitative analysis of the bile acid in the serum sample can be realized, and the method has good application prospect in the field of the quantitative analysis of the serum determination.
Drawings
FIG. 1A fitted curve of ursodeoxycholic acid of example 1;
FIG. 2 cholic acid mass spectrum of example 1;
FIG. 3A mass spectrum of taurocholate of example 1;
FIG. 4A is a mass spectrum of procholic acid in example 1;
FIG. 5A is a mass spectrum of chenodeoxycholic acid in example 1;
FIG. 6A spectrum of hyodeoxycholic acid from example 1;
FIG. 7 mass spectrum of ursodeoxycholic acid of example 1.
Detailed Description
The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples of the present invention, unless otherwise specified, was performed using conventional testing methods in the art. It should be understood that the terminology used in the description of the embodiments of the invention presented is for the purpose of describing particular embodiments only, and is not intended to be limiting of the disclosure of the embodiments of the invention.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong; other test methods and techniques not specifically identified in the examples of the present invention are those generally employed by those skilled in the art.
The terms "substantially" and "about" are used herein to describe small fluctuations. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Numerical data presented or represented herein in a range format is used only for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range. For example, a numerical range of "1 to 5%" should be interpreted to include not only the explicitly recited values of 1% to 5%, but also include individual values and sub-ranges within the indicated range. Thus, individual values, such as 2%, 3.5% and 4%, and subranges, such as 1% to 3%, 2% to 4% and 3% to 5%, etc., are included in this numerical range. The same principle applies to ranges reciting only one numerical value. Moreover, such an interpretation applies regardless of the breadth of the range or the characteristics being described.
In this document, including the claims, conjunctions such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "and the like are to be construed as open-ended, i.e., to mean" including, but not limited to. Only the conjunctions "consisting of … …" and "consisting of … …" are closed conjunctions.
Numerous specific details are set forth in the following examples in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
On the premise of no conflict, the technical features disclosed by the embodiment of the invention can be combined at will, and the obtained technical scheme belongs to the disclosure of the embodiment of the invention.
In some embodiments, the absolute quantitative analysis method of bile acid in serum sample comprises:
preparing a working reagent which is a solution containing isotope labels of cholic acid-D4, lithocholic acid-D4 and ursodeoxycholic acid-2, 4-D4;
preparing a sample to be tested by using the prepared working reagent and a serum sample;
configuring at least two calibration reagents; the targeting agent is a solution comprising cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, tauchenodeoxycholic acid, deoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, lithocholic acid, glucolithocholic acid, taurocholate, hyocholic acid, gan Qing cholic acid, taurocholate, alpha-murine cholic acid, taurine-alpha-polyphenol sodium salt, beta-murine cholic acid, omega-murine cholic acid, tauro-omega-murine cholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, sodium taurochenodeoxycholic acid, 5 beta-cholic acid-3 alpha, 6 beta-diol, dehydrocholic acid, glycodehydrocholic acid, taurodeoxycholic acid, 7-keto-3 alpha, 12-alpha-dihydroxycholic acid, isodeoxycholic acid, orthocholic acid, 5-beta-cholic acid-3-alpha-alcohol-6-one, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, 12-ketolithocholic acid,12 ketochenodeoxycholic acid, nordeoxycholic acid, and isolithocholic acid as a solute; the concentration of each solute in the calibration reagent is the same, and the concentrations of at least two calibration reagents are different;
determining a calibration reagent by utilizing high performance liquid chromatography-tandem mass spectrometry, and determining a fitting curve of each solute;
and determining the absolute content of bile acid in the sample to be detected by utilizing high performance liquid chromatography-tandem mass spectrometry according to the determination result and the obtained fitting curve.
Typically, the scaling reagent includes thirty-eight bile acids, as solutes of the scaling reagent, cholic Acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), chenodeoxycholic acid (chenodeoxycholic acid, CDCA), taurochenodeoxycholic acid (TCDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), taurochenoxycholic acid (taurodeoxycholic acid, TDCA), ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (glycoursodeoxycholic acid, GUDCA), taurocholodeoxycholic acid (turdca), lithocholic acid (lithocholic acid, LCA), glucocholic acid (glycocholic acid, GLCA), taurocholic acid (taurolithocholic acid, TLCA), hyocholic acid (HCA), gan Qing cholic acid (glycohyocholic acid, GHCA), taurocholate (taurohyocholic acid, THCA), alpha-murine acid (alpha-muricholic acid, alpha MCA), taurine-alpha-polyphenol sodium salt (tauro-alpha-murichlic acid, T alpha MCA), beta-murine acid (beta-muricholic acid, beta MCA), omega-murine acid (omega-muricholic acid, omega MCA), tauro-omega-murine acid (tauro-omega-muricholic acid, T omega MCA), hyodeoxycholic acid (hyodeoxycholic acid, HDCA), glycohyodeoxycholic acid (glycohyodeoxycholic acid, GHDCA), taurochenoxysodium (taurohyodeoxycholic acid, THDCA), 5 beta-3 alpha, 6 beta-diol (Murocholic Acid, muro-CA), dehydrocholic Acid (dehydrocholic Acid, DHCA), glycodehydrocholic Acid (glycodehydrocholic Acid, GDHCA), taurocholic Acid (taurodehydrocholic Acid, TDHCA), 7-keto-3α,12- α -dihydroxycholic Acid (7-ketodeoxycholic Acid, 7-KDCA), isodeoxycholic Acid (isodeoxycholic Acid, isoDCA), orthocholic Acid (apocholic Acid, ACA), 5-beta-cholic Acid-3- α -alcohol-6-one (6-ketolithocholic Acid, 6-KLCA), 3α -hydroxy-7-oxo-5β -cholanic Acid (7-ketolithocholic Acid, 7-KLCA), 12-ketolithocholic Acid (12-ketolithocholic Acid, 12-KLCA), 12-ketochenodeoxycholic Acid (12-ketochenodeoxycholic Acid, 12-KCDCA), nordeoxycholic Acid (23-nordeoxycholic Acid, 23-NDCA) and isolithocholic Acid (isosorbic Acid, isostearic Acid), which are used as a calibration curve for the bile Acid in a liquid phase, and a mass spectrometry system to further quantitatively analyze bile Acid pairs in a bile phase system to determine the bile Acid calibration curve; by using the measured fitting curve, absolute quantitative analysis can be performed on all bile acids contained in the sample to be measured.
The concentration of each component in the targeting agent is typically the same, i.e., the targeting agent comprises each bile acid in the same concentration.
In the general absolute quantitative analysis method of bile acid, at least two calibration reagents are adopted to perform calibration detection on bile acid, a fitting curve of the bile acid is determined, the more the types of the calibration reagents are, the higher the accuracy of the determined fitting curve is, the higher the reproducibility is, and the accuracy of determining the quantitative analysis result is improved. Too many types of calibration reagents can reduce the efficiency of the assay and increase the cost of the assay. Under normal conditions, a reasonable number of calibration reagent types are selected, and the detection accuracy requirement is met. A number of calibration reagents are typically selected, each of which has a different concentration, to process the test results to determine a fitted curve.
The use of isotopic labels of analytes as internal standards in working reagents generally improves reproducibility and stability of the analysis results. Is favorable for determining and quantitatively analyzing bile acid.
In some embodiments, the solvent for the working agent is methanol.
In some embodiments, the solvent of the sample to be tested is acetonitrile or isopropanol.
In some embodiments, the solvent of the targeting agent is methanol.
In some embodiments, a method for configuring a sample to be tested includes:
uniformly mixing a working reagent, a serum sample and a solvent according to a set proportion;
after mixing evenly, centrifugal precipitation is carried out, supernatant fluid is taken, and nitrogen purging is utilized for drying;
and re-dissolving the sample by using a re-dissolving solvent, and taking the supernatant after re-dissolving to obtain a sample to be tested.
In some embodiments, a method for configuring a sample to be tested includes:
uniformly mixing a working reagent, a serum sample and a solvent according to a set proportion;
incubating for 20 minutes at 4 ℃;
centrifuging 11000g at 4 ℃ for 20 minutes, collecting supernatant, and drying by nitrogen purging;
and re-dissolving the sample by using a re-dissolving solvent, and taking the supernatant after re-dissolving to obtain a sample to be tested.
In some embodiments, the method of uniformly mixing the working reagent, serum sample, and solvent comprises: the pipetting device is used for blowing, reversing and mixing, rotating and mixing or vortex shaking and mixing.
In some preferred embodiments, vortex shaking is used for mixing for 15-30 seconds.
The absolute quantitative analysis method of bile acid in serum samples disclosed in some embodiments comprises the steps that a redissolution solvent is an acetonitrile aqueous solution of formic acid with the mass content of 0.1% and/or a methanol aqueous solution with the mass content of 60%, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1.
in some embodiments, the volume ratio of the working reagent, the serum sample and the solvent in the sample to be measured is 3:47:200.
The absolute quantitative analysis method of bile acid in serum samples disclosed in some embodiments can be applied to other body fluid environments of human body for detecting bile acid, such as cerebrospinal fluid and urine.
In some embodiments, the sample to be tested and the calibration reagent are batched for high performance liquid chromatography-tandem mass spectrometry system detection; the detection comprises the following steps:
a mobile phase reagent is adopted to balance a high performance liquid chromatography system;
connecting the chromatographic column to a high performance liquid chromatography system;
sampling the calibration reagent in sequence from low concentration to high concentration; obtaining a calibration detection result;
and carrying out sample injection on the obtained sample to be detected to obtain a detection result of the sample to be detected.
The absolute quantitative analysis method of bile acid in serum sample disclosed in some embodiments, the test conditions of high performance liquid chromatography-tandem mass spectrometry include:
conditions of high performance liquid chromatography:
mobile phase a:0.1% formic acid in water, mobile phase B:0.1% formic acid in acetonitrile;
the separation conditions are listed in table 2;
TABLE 2 list of conditions for high performance liquid chromatography
The tandem mass spectrum is formed by connecting three four-level rod mass spectrometers in series, and the detection conditions are as follows:
ionization mode: electrospray ionization, ESI (-;
ion spray voltage: 2.7kV;
sheath gas (Arb): 50;
auxiliary device (Arb): 10;
purge gas (Arb): 1, a step of;
ion transport capillary temperature: 320 ℃;
sprayer temperature: 150 ℃;
the detection mode is as follows: selecting reaction monitoring;
cycle time: 1.0sec;
q1 resolution: 0.7;
q3 resolution: 0.7;
collision gas: 1.5mTorr;
in-source cleavage voltage: 0V;
chromatographic peak width: 12sec.
Some embodiments disclose methods for absolute quantitative analysis of bile acids in serum samples, the determination of the fitted curve comprising:
according to the detection result of the calibration reagent, counting the area of each solute mass spectrum peak in the calibration reagent;
carrying out normalization treatment according to the base peaks to obtain the ionic strength of each solute;
and linearly fitting the ionic strength of each solute with the corresponding ionic concentration to obtain a fitted curve model.
In some embodiments, using the test results and the fitted curve of the sample to be tested, absolute quantitative analysis of bile acid comprises:
according to the mass spectrum detection result of the sample to be detected, counting the mass spectrum peak area of each bile acid component in the sample to be detected;
carrying out normalization treatment according to the basic peak to obtain the ionic strength of each bile acid component;
and respectively bringing the ionic strength of each bile acid component into a fitting curve to obtain the corresponding molecular number.
In some embodiments, at least one column is used in high performance liquid chromatography, including at least one reverse-phase column.
In some embodiments, tandem mass spectrometry systems employ electrospray ion sources.
In some embodiments, the tandem mass spectrometry system is a quadrupole tandem mass spectrometry system.
In some embodiments, the detection method of the tandem mass spectrometry system is a multi-reaction channel detection, including at least two of a parent ion scan, a child ion scan, and a neutral loss scan.
Further exemplary details are described below in connection with the embodiments.
Example 1
Example 1 discloses a method for absolute quantitative analysis of bile acids in blood serum samples. The method specifically comprises the following steps:
collecting 50 μl of serum samples;
preparing a working reagent which is a solution containing isotope labels of cholic acid-D4, lithocholic acid-D4 and ursodeoxycholic acid-2, 4-D4; the composition and concentration of the working agent are listed in table 3;
TABLE 3 working reagent composition and concentration list
Preparing a sample to be tested by using the prepared working reagent and a serum sample; the components and proportions of the sample to be tested are shown in Table 4;
TABLE 4 composition and volume list of samples to be tested
Serum samples, working reagent and acetonitrile were mixed in the volumes of table 2 and vortexed for 30 seconds to thoroughly mix.
The uniformly mixed sample to be tested is incubated for 20 minutes at 4 ℃, then centrifuged for 20 minutes at 11000g at 4 ℃, and the supernatant is taken out and placed in a new centrifuge tube. Placing the supernatant on a nitrogen blowing instrument, performing nitrogen blowing drying, re-dissolving with 50 μl of 60% methanol water, simply centrifuging, transferring into a sample bottle, and placing on machine or storing at-20deg.C for about 2 weeks or at-80deg.C for 3 months;
preparing thirteen calibration reagents; the targeting agent is a solution comprising cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, tauchenodeoxycholic acid, deoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, lithocholic acid, glucolithocholic acid, taurocholate, hyocholic acid, gan Qing cholic acid, taurocholate, alpha-murine cholic acid, taurine-alpha-polyphenol sodium salt, beta-murine cholic acid, omega-murine cholic acid, tauro-omega-murine cholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, sodium taurochenodeoxycholic acid, 5 beta-cholic acid-3 alpha, 6 beta-diol, dehydrocholic acid, glycodehydrocholic acid, taurodeoxycholic acid, 7-keto-3 alpha, 12-alpha-dihydroxycholic acid, isodeoxycholic acid, orthocholic acid, 5-beta-cholic acid-3-alpha-alcohol-6-one, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, 12-ketolithocholic acid,12 ketochenodeoxycholic acid, nordeoxycholic acid, and isostonic acid as a solute; the concentration of each solute in the calibration reagent is the same; thirteen calibration reagents are denoted as C1, C2, … …, C13, respectively, and the thirteen calibration reagents and corresponding concentrations are listed in Table 5;
in Table 5, the first column shows thirteen calibration reagents and the second column shows the concentration of each component in each calibration reagent.
Table 5 example 1 calibration reagents and concentration lists thereof
Determining a calibration reagent by utilizing high performance liquid chromatography-tandem mass spectrometry, and determining a fitting curve of each solute;
first, vanquish high performance liquid chromatography was equilibrated at a flow rate of 3.00 mL/min for 5min using a mobile phase reagent, mobile phase reagent A was an aqueous solution of 0.1% formic acid, mobile phase reagent B was an acetonitrile solution of 0.1% formic acid, and the specific compositions and concentrations are shown in Table 6;
TABLE 6 composition and concentration of mobile phase reagent A, B
The column ACQUITY BEH C18 column (1.7 μm, 100 mm ×2.1 mm internal dimensions) (Waters, milford, mass.) was then connected to a high performance liquid chromatograph and after maintaining a constant temperature of 45℃the split sample was started.
The mobile phase of the high performance liquid chromatography and the separation conditions are shown in Table 7.
TABLE 7 high performance liquid chromatography mobile phase and separation conditions
And then performing multi-reaction detection scanning by a tandem mass spectrometer formed by connecting three four-stage rod mass spectrometers in series, wherein the detection conditions of the mass spectrum are as follows:
ionization mode: electrospray ionization, ESI (-;
ion spray voltage: 2.7kV;
sheath gas (Arb): 50;
auxiliary device (Arb): 10;
purge gas (Arb): 1, a step of;
ion transport capillary temperature: 320 ℃;
sprayer temperature: 150 ℃;
the detection mode is as follows: selecting reaction monitoring;
cycle time: 1.0sec;
q1 resolution: 0.7;
q3 resolution: 0.7;
collision gas: 1.5mTorr;
in-source cleavage voltage: 0V;
chromatographic peak width: 12sec.
Thirteen calibration reagents are sequentially injected into the high performance liquid chromatography-tandem mass spectrometry system from low concentration to high concentration for analysis; and detecting the abundance of fragment ions of the thirteen scanned calibration reagents, and calibrating according to the mass spectrum detection result of the thirteen calibration reagents to determine a fitting curve.
Firstly, counting the area of mass spectrum peaks of bile acid of each solute component in a calibration reagent according to a fragment ion abundance diagram of the calibration reagent;
carrying out normalization treatment according to the basic peaks to respectively obtain the ionic strength of each bile acid at different concentrations;
finally, the ionic strength of each component is linearly fitted to its corresponding ionic concentration.
Since the bile acid in the calibration reagent in this example 1 belongs to a class of bile acid substances, the detected bile acid is of a large variety, but the same linear fitting method can be adopted to obtain the fitting curve of each bile acid.
Fig. 1 shows a linear curve model of one of the bile acids, ursodeoxycholic acid (ursodeoxycholic acid, UDCA), after fitting, in fig. 1, the horizontal axis represents concentration, the vertical axis represents the ratio of the compound peak area to the internal standard peak area, and the fitting parameters are: k=0.10877, b= 1.32625e -4 The fitting coefficient was 0.9968.
The fitted curve is expressed as:
Y=0.000132625+0.10877•X
according to the inspection and analysis process of the thirteen calibration reagents, detecting and analyzing the sample to be detected in the same high performance liquid chromatography and tandem mass spectrometry system; and quantitatively analyzing the bile acid in the sample to be detected by utilizing the obtained bile acid fitting curve according to the detection result of the bile acid in the sample to be detected. The ionic signal intensity of different bile acids is brought into a corresponding fitting curve model, so that the analysis quantity of the bile acids with different carbon chain lengths in a serum sample can be calculated.
Illustratively, fig. 2 illustrates a mass spectrum of Cholic Acid (CA), fig. 3 illustrates a mass spectrum of taurocholic acid (TCA), fig. 4 illustrates a mass spectrum of raw cholic acid (ACA), fig. 5 illustrates a mass spectrum of chenodeoxycholic acid (CDCA), fig. 6 illustrates hyodeoxycholic acid (HDCA), and fig. 7 illustrates a mass spectrum of ursodeoxycholic acid (UDCA).
According to the absolute quantitative analysis method for bile acid in the serum sample disclosed by the embodiment of the invention, the isotope label containing the object to be detected is used as an internal standard in the working reagent, so that the stability and repeatability of an analysis result are ensured, different components of the bile acid are effectively separated through the designed mobile phase chromatographic conditions, the mutual interference among different components is reduced, the interference of a solvent and a mobile phase is reduced, and the reliability of specificity and quantitative detection is ensured; the sensitivity and the specificity of the analysis are ensured through the tandem mass spectrum, the serum sample with low sample quantity is utilized by using the calibration reagent with different components and concentrations, the absolute quantitative analysis of the bile acid in the serum sample can be realized, and the method has good application prospect in the field of the quantitative analysis of the serum determination.
The technical solutions disclosed in the embodiments of the present invention and the technical details disclosed in the embodiments of the present invention are only exemplary to illustrate the inventive concept of the present invention, and do not constitute a limitation on the technical solutions of the embodiments of the present invention, and all conventional changes, substitutions or combinations of the technical details disclosed in the embodiments of the present invention have the same inventive concept as the present invention, and are within the scope of the claims of the present invention.
Claims (10)
1. A method for absolute quantitative analysis of bile acids in a serum sample, comprising:
preparing a working reagent which is a solution containing isotope labels cholic acid-D4, lithocholic acid-D4 and ursodeoxycholic acid-2, 4-D4;
preparing a sample to be tested by using the prepared working reagent and a serum sample;
configuring at least two calibration reagents; the targeting agent is a solution comprising cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, tauchenodeoxycholic acid, deoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, lithocholic acid, glucolithocholic acid, taurocholic acid, hyocholic acid, gan Qing cholic acid, taurocholic acid, alpha-murine cholic acid, taurine-alpha-polyphenol sodium salt, beta-murine cholic acid, omega-murine cholic acid, tauro-omega-cholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, sodium tausodeoxycholic acid, 5 beta-cholic acid-3 alpha, 6 beta-diol, dehydrocholic acid, aminodehydrocholic acid, tauro-dehydrocholic acid, 7-one-3 alpha, 12-alpha-dihydroxycholic acid, isodeoxycholic acid, orthocholic acid, 5-beta-cholic acid-3-alpha-alcohol-6-one, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, 12-ketocholic acid, 12-one chenodeoxycholic acid, nordeoxycholic acid, and isolithocholic acid as a solute; the concentration of each solute in the calibration reagent is the same, and the concentration of at least two calibration reagents is different;
determining a calibration reagent by utilizing high performance liquid chromatography-tandem mass spectrometry, and determining a fitting curve of each solute;
and determining the absolute content of bile acid in the sample to be detected by utilizing high performance liquid chromatography-tandem mass spectrometry according to the determination result and the obtained fitting curve.
2. The method of claim 1, wherein the solvent of the working reagent is methanol.
3. The method according to claim 1, wherein the solvent of the sample to be tested is acetonitrile or isopropanol.
4. The method of claim 1, wherein the solvent for the calibration reagent is methanol.
5. The method for absolute quantitative analysis of bile acid in a serum sample according to claim 1, wherein the method for configuring the sample to be tested comprises:
uniformly mixing a working reagent, a serum sample and a solvent according to a set proportion;
after mixing evenly, centrifugal precipitation is carried out, supernatant fluid is taken, and nitrogen purging is utilized for drying;
and re-dissolving the sample by using a re-dissolving solvent, and taking the supernatant after re-dissolving to obtain a sample to be tested.
6. The method according to claim 5, wherein the redissolving solvent is an acetonitrile aqueous solution of formic acid with a mass content of 0.1% and/or a methanol aqueous solution with a mass content of 60%, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1.
7. the method according to claim 5, wherein the volume ratio of the working reagent, the serum sample and the solvent in the sample to be measured is 3:47:200.
8. The method for absolute quantitative analysis of bile acid in a serum sample according to claim 1, wherein the test conditions of the high performance liquid chromatography-tandem mass spectrometry include:
conditions of high performance liquid chromatography:
mobile phase a:0.1% formic acid in water, mobile phase B:0.1% formic acid in acetonitrile;
separation conditions:
the tandem mass spectrum is formed by connecting three four-level rod mass spectrometers in series, and the detection conditions are as follows:
ionization mode: electrospray ionization, ESI (-;
ion spray voltage: 2.7kV;
sheath gas (Arb): 50;
auxiliary device (Arb): 10;
purge gas (Arb): 1, a step of;
ion transport capillary temperature: 320 ℃;
sprayer temperature: 150 ℃;
the detection mode is as follows: selecting reaction monitoring;
cycle time: 1.0sec;
q1 resolution: 0.7;
q3 resolution: 0.7;
collision gas: 1.5mTorr;
in-source cleavage voltage: 0V;
chromatographic peak width: 12sec.
9. The method of claim 1, wherein the determination of the fitted curve comprises:
according to the detection result of the calibration reagent, counting the area of each solute mass spectrum peak in the calibration reagent;
carrying out normalization treatment according to the base peaks to obtain the ionic strength of each solute;
and linearly fitting the ionic strength of each solute with the corresponding ionic concentration to obtain a fitted curve model.
10. The method for absolute quantitative analysis of bile acid in serum sample according to claim 1, wherein the absolute quantitative analysis of bile acid by using the test result and the fitted curve of the sample to be tested comprises:
according to the mass spectrum detection result of the sample to be detected, counting the mass spectrum peak area of each bile acid component in the sample to be detected;
carrying out normalization treatment according to the basic peak to obtain the ionic strength of each bile acid component;
and respectively bringing the ionic strength of each bile acid component into a fitting curve to obtain the corresponding molecular number.
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