CN110596295A - Method for detecting bile acid - Google Patents
Method for detecting bile acid Download PDFInfo
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- CN110596295A CN110596295A CN201911000008.7A CN201911000008A CN110596295A CN 110596295 A CN110596295 A CN 110596295A CN 201911000008 A CN201911000008 A CN 201911000008A CN 110596295 A CN110596295 A CN 110596295A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
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Abstract
The invention discloses a method for detecting bile acid, which comprises the following steps: 1) preparing a standard substance; 2) ultra-high performance liquid chromatography separation: carrying out chromatographic separation on bile acid on a liquid chromatographic column; 3) mass spectrometry was performed in parallel reaction monitoring mode: the bile acid after chromatographic separation enters a mass spectrometer for detection, and the detection limit and the quantification limit of the method are calculated according to the signal-to-noise ratio; 4) and (3) drawing a calibration curve: performing regression analysis by using a least square method, and drawing a calibration curve; 5) detection of bile acids in samples: carrying out sample loading and analysis on the sample by adopting the steps 2) and 3) to obtain a ratio of the bile acid in the sample to the internal standard, substituting the ratio into the calibration curve, and calculating the type and the content of the bile acid in the sample; the present invention provides a method that can detect more bile acids at a time.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting bile acid.
Background
Bile acid is a very important component in the human body, and especially plays a great role in the process of fat metabolism. Bile acid is the main component in bile, the generation and metabolism of bile acid is closely related to liver, liver cells are diseased, and serum total bile acid changes along with the change. However, the specificity of total bile acid for diagnosing different diseases is poor, and the guiding effect of the detection result on the diagnosis and treatment of the diseases is limited. Total bile acid is a generic term for a plurality of structurally similar bile acids of different subtypes, wherein one type of hydrophobic bile acid is toxic and can destroy liver cells, while the other type of hydrophilic bile acid has the toxic effect of antagonizing the hydrophobic bile acid, can stimulate the secretion of hepatobiliary ducts and can regulate immunity, and is often used for treating ICP clinically, and the increase of the total bile acid alone cannot judge whether the treatment is needed. At present, the accurate medical diagnosis (research) center of the clinical laboratory of hospitals can detect 15 different subtypes of bile acids at one time by a liquid chromatography-mass spectrometry combined analysis technology, and has very important significance for clinical diagnosis and treatment of ICP and other liver diseases.
However, the existing methods for detecting bile acid are complex, and basically all the methods are used for quantifying total bile acid or only detecting partial bile acid, so the detection result is not comprehensive.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting more kinds of bile acid at one time and a wide range of detection samples, which comprises the following steps:
1) preparing a standard substance;
2) ultra-high performance liquid chromatography separation: carrying out chromatographic separation on bile acid on a liquid chromatographic column;
3) mass spectrometry was performed in parallel reaction monitoring mode: the bile acid after chromatographic separation enters a mass spectrometer for detection, and the detection limit and the quantification limit of the method are calculated according to the signal-to-noise ratio;
4) and (3) drawing a calibration curve: performing regression analysis by using a least square method, and drawing a calibration curve;
5) detection of bile acids in samples: and (3) carrying out sampling and analysis on the sample by adopting the steps 2) and 3) to obtain the ratio of the bile acid in the sample to the internal standard, substituting the ratio into the calibration curve, and calculating the type and the content of the bile acid in the sample.
Further, the conditions of the ultra-high performance liquid chromatography in the step 2) are as follows: the chromatographic specification of C18 is 150 × 2.1mm,1.7 μm, liquid chromatogram A phase is 0.01% formic acid water solution, phase B is acetonitrile, column incubator temperature is 40 deg.C, sample tray is set at 4 deg.C, and injection volume is 3 μ L.
Further, the conditions of mass spectrometry in the step 3) are as follows: spray voltage +3500/-3100V, shear gas (N2) flow rate 40, Aux gas (N2) flow rate 15, Sweep gas (N2) flow rate 0, Aux gas (N2) temperature 350 ℃, and caliper temperature 320 ℃.
Further, the parameters of the detection limit and the quantification limit of the method in the step 3) are set as follows: the method minimum detection limit is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 3, and the method minimum quantitation limit is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 10.
Further, the parameters for drawing the calibration curve in the step 4) are set as follows: and (3) performing regression analysis by using a least square method, wherein when the weight is set to be 1/x, the recovery rate and the correlation coefficient of the calibration solution are best, and if the signal-to-noise ratio (S/N) of a certain calibration concentration is close to or less than 20 or the recovery rate exceeds the range of 80-120%, the concentration calibration point is excluded.
Further, the method further comprises: detecting the precision and accuracy of the method, wherein the precision of the method is evaluated by the standard relative deviation of the repeated sample injection of the QC sample; the accuracy is evaluated by the standard recovery rate of the QC sample, and the percentage value of the measured concentration and the standard concentration is the standard recovery rate.
Further, the sample comprises serum, plasma, feces, liver tissue, intestinal contents, bile, gastric juice.
Compared with the prior art, the technical scheme of the invention has the following advantages: the invention has successfully carried out the quantitative detection of bile acid on samples such as serum, feces, bile and the like, and has the advantages of low sample amount, quick detection time, accurate quantitative result, various types of detected bile acid and very wide application prospect in the clinical field.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the results of detection of bile acid in the standard substance in example 1;
FIG. 2 shows the results of detection of bile acid in the standard substance in example 1;
FIG. 3 shows the results of detection of bile acid in the standard substance in example 1;
FIG. 4 shows the results of detection of bile acid in the standard substance in example 1;
FIG. 5 shows the results of bile acid detection of human stool samples in example 3;
FIG. 6 shows the results of bile acid detection of human stool samples in example 3;
FIG. 7 shows the results of bile acid detection of human stool samples in example 3;
FIG. 8 shows the results of bile acid detection of human stool samples in example 3;
FIG. 9 is a calibration curve for α -murine cholic acid;
FIG. 10 is a calibration curve for glycodehydrocholic acid;
FIG. 11 is a calibration curve for tauroursodeoxycholic acid.
Detailed Description
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
EXAMPLE 1 detection of standards
The bile acid in the standard was detected in the following manner:
1. standard preparation
Accurately weighing corresponding amount of standard substance in a 10mL volumetric flask, and respectively preparing into 10mmol/L standard substance stock solutions. And (3) taking a corresponding amount of standard substance stock solution into a 10mL volumetric flask to prepare a mixed standard solution. The standard solutions were diluted sequentially to give a series of calibration solutions.
2. Detection on machine
2.1 mobile phase conditions: the target compound was chromatographed on a Waters acquisition UPLC BEH C18(150 × 2.1mm,1.7 μm, Waters) liquid chromatography column using Agilent 1290Infinity series (Agilent Technologies) hplc. The liquid chromatogram is characterized in that the phase A is 0.01 percent formic acid water solution, the phase B is acetonitrile, and the chromatogram gradient is shown in Table S2. The column oven temperature was 40 ℃, the sample plate set at 4 ℃ and the injection volume was 3 μ L.
2.2 Mass Spectrometry conditions: the project uses a Q active Focus high-resolution mass spectrometer to perform mass spectrometry in a Parallel Reaction Monitoring (PRM) mode. The ion source parameters were as follows: sprayvoltage +3500/-3100V, Sheath gas (N2) flow rate 40, Aux gas (N2) flow rate 15, Sweep gas (N2) flow rate 0, Aux gas (N2) temperature 350 ℃, and calillary temperature 320 ℃.
Standard solutions of the target compounds were introduced into the mass spectra before UHPLC-MS/MS analysis. For each target compound, optimizing its PRM parameter; since most of the bile acid compounds have daughter ion spectra in which daughter ions having an intensity sufficient for quantification are not present, parent ions under high-resolution conditions are selected for quantification.
3. Calibration curve
The calibration solutions were subjected to UPLC-PRM-MS/MS analysis using the methods described previously. Wherein the calibration curve of a part of bile acid is shown in FIGS. 9 to 11, y represents the peak area of the target compound, and x represents the concentration (nmol/L) of the target compound. The regression analysis was performed using the least squares method, and the calibration solution recovery (accuracy) and correlation coefficient (R2) were best when the weight was set to 1/x. If the signal-to-noise ratio (S/N) of a certain calibration concentration is close to or less than 20, or the recovery rate exceeds the range of 80-120%, the concentration calibration point is excluded.
4. Method detection limit and quantification limit
And sequentially diluting the calibration solution by 2 times, performing UHPLC-PRM-MS analysis, and calculating the detection limit and the quantification limit of the method according to the signal-to-noise ratio of the calibration solution. The method minimum detection limit (LLOD) is defined as the concentration of the compound for a signal-to-noise ratio of 3, and the method minimum quantitation limit (LLOQ) is defined as the concentration of the compound for a signal-to-noise ratio of 10.
5. Method precision and accuracy
The precision of the method was assessed by the standard relative deviation (RSD) of QC sample replicate injections. The accuracy was assessed by the Recovery (Recovery) of the QC sample, and the percentage value of the measured concentration to the spiked concentration was the spiked Recovery.
6. Results of the experiment
Standard solutions and sample Extraction Ion Chromatograms (EICs) are shown in fig. 1-4, from which it can be seen that:
1) according to the analysis method adopted by the project, all target compounds show better chromatographic peaks;
2) and the chromatographic separation of each target compound is well realized. The spectral peaks in the figure are as follows: (1) dehydrolithocholic acid (Dehydrolithocholic acid), (2) Allolithocholic acid (Allolithocholic acid), (3) isocratic acid (isocoholic acid), (4) Lithocholic acid (Lithocholic acid), (5), 23-demethoxycholic acid (23-Nordeoxycholic acid), (6), 7-Ketolithocholic acid (7-Ketolithocholic acid), (7, 12-Ketolithocholic acid (12-Ketolithocholic acid), (8) protopholic acid (Apocholic acid), (9), Ursodeoxycholic acid, (10), Hyodeoxycholic acid, (11), Chenodeoxycholic acid (Chenodeoxycholic acid), (12), Deoxycholic acid (Deoxycholic acid), (13, isocoholic acid), (14, 12, 7-Lithocholic acid), (7,12, 7-Lithocholic acid), 7-diketolithocholic acid (6,7-diketolithocholic acid), (17), 7-Ketodeoxycholic acid (7-Ketodeoxycholic acid), (18), 12-Dehydrocholic acid (12-Dehydrocholic acid), (19), 3-Dehydrocholic acid (3-Dehydrocholic acid), (20), ursolic acid (Ursocholic acid), (21), α -murine Cholic acid (α -Muricholic acid), (22), β -murine Cholic acid (β -Muricholic acid), (23), λ -murine Cholic acid (λ -Muricholic acid), (24), Allocholic acid, (25), Cholic acid (Cholic acid), (26), Glycolithocholic acid (Glycodeoxycholic acid), (27), Glycodeoxycholic acid (Glycodeoxycholic acid), (30), (31) glycodehydrocholic acid, (32) Glyco- λ -murine cholic acid, (33) Glycocholic acid (Glycocholic acid), (34) taurocholic acid (Taurolithocholic acid), (35) Tauroursodeoxycholic acid (Tauroursodeoxycholic acid), (36) Taurochenodeoxycholic acid (tauurodeoxycholic acid), (37) Taurodeoxycholic acid (Taurochenodeoxycholic acid), (38) Taurodeoxycholic acid (Taurodeoxycholic acid).
3) The quantitative parameters of the target compound are shown in the specification, wherein the lowest detection limit (LLODs) is between 0.24 and 0.98nmol/L, and the lowest quantitative limit (LLOQs) is between 0.49 and 3.91 nmol/L; can meet the requirement of targeted metabonomics analysis; the recovery rate (recovery) and the standard relative deviation (RSD) of the QC sample are shown in Table S5, and the repeated sample injection times of the QC sample are 6 times; the average recovery of all target compounds is between 80.2% and 100.8%, and the standard relative deviation is less than 10.6%. The data show that the method can accurately and reliably detect the content of the target metabolite in the sample.
Example 2
Detection of bile acids in mouse serum samples:
first, treatment of mouse serum samples
1. Adding 100 μ L mouse serum into an EP tube, adding 400 μ L extractive solution (methanol: acetonitrile: 1(V/V) containing internal standard 12.5nmol/L), vortexing for 30s, and mixing;
2. ultrasonic treatment for 5min (ice water bath);
3. standing at-20 ℃ for 1 h;
4. centrifuging the sample at 4 ℃ and 12000rpm for 15 min;
5. 400 μ L of the supernatant was taken in an EP tube, blown dry with nitrogen, and 80 μ L of a 1% formic acid solution (methanol: acetonitrile: water ═ 2: 2: 1(V/V)) was redissolved.
6. Mixing by vortex for 30s, and performing ultrasonic treatment for 5min (ice water bath);
7. centrifuging at 12000rpm for 15min at 4 deg.C, collecting supernatant to LC sample bottle for UHPLC-MS/MS analysis.
Secondly, detecting bile acid
Bile acid was detected in a mouse serum sample by the method of example 1.
Three, result in
Detecting and analyzing the target compounds of bile acids in the sample, and detecting a total of 28 bile acid compounds in the sample
Example 3
Detection of bile acids in human fecal samples:
human fecal sample treatment
1. Weighing 50mg of sample, adding 1000 mu L of 1% formic acid extract (methanol: acetonitrile: water: 2: 1, containing internal standard 50nmol/L), and mixing by vortex for 30 s;
2. adding steel balls, grinding at 45Hz for 4min, and performing ultrasonic treatment for 5min (ice water bath);
3. repeating the step 2 and 3 times;
4. standing at-20 ℃ for 1 h;
5. centrifuging the sample at 4 ℃ at 12000rpm for 15 min; and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
Secondly, detecting bile acid
Bile acids in human fecal samples were detected using the method of example 1.
Three, result in
The analysis and detection of 40 human fecal samples showed that 35 total bile acid compounds were detected in the samples as shown in FIGS. 5-8.
Example 4
Treatment of serum, bile and liver samples of pelteobagrus fulvidraco
A. Serum/bile samples:
1. adding 100 μ L of serum/bile sample into EP tube, adding 400 μ L of extractive solution (methanol: acetonitrile 1: 1, containing 1% formic acid), vortex for 30s, and mixing;
2. ultrasonic treatment for 5min (ice water bath);
3. standing at-20 ℃ for 1 h;
4. centrifuging the sample at 4 ℃ and 12000rpm for 15 min;
5. 400 μ L of the supernatant was taken in an EP tube, blown dry with nitrogen, and 80 μ L of 1% formic acid solution (methanol: acetonitrile: water: 2: 1) was redissolved.
6. Mixing by vortex for 30s, and performing ultrasonic treatment for 5min (ice water bath);
7. centrifuging at 12000rpm for 15min at 4 deg.C, collecting supernatant to LC sample bottle for UHPLC-MS/MS analysis.
B. Liver samples:
1. weighing 50mg of liver sample, adding 1000 mu L of 1% formic acid extract (methanol: acetonitrile: water: 2: 1), and mixing by vortex for 30 s;
2. adding steel balls, grinding at 45Hz for 4min, and performing ultrasonic treatment for 5min (ice water bath);
3. repeating the step 2 and 3 times;
4. standing at-20 ℃ for 1 h;
5. centrifuging the sample at 4 ℃ and 12000rpm for 15 min; and taking the supernatant to an LC sample injection bottle for UHPLC-MS/MS analysis.
Secondly, detecting bile acid
The method of example 1 is adopted to detect bile acid in serum, bile and liver samples of the pelteobagrus fulvidraco.
Three, result in
Aiming at 12 pelteobagrus fulvidraco serum samples, 12 bile pelteobagrus fulvidraco samples and 12 pelteobagrus fulvidraco bile samples, the total analysis and detection of 36 samples are carried out, and 27 kinds of bile acids are detected in the samples.
In conclusion, bile acid has very important clinical significance, and the bile acid ratio can reflect the activity of the enzyme related to the liver and intestinal flora.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (7)
1. A method for detecting bile acid, comprising the steps of:
1) preparing a standard substance;
2) ultra-high performance liquid chromatography separation: carrying out chromatographic separation on bile acid on a liquid chromatographic column;
3) mass spectrometry was performed in parallel reaction monitoring mode: the bile acid after chromatographic separation enters a mass spectrometer for detection, and the detection limit and the quantification limit of the method are calculated according to the signal-to-noise ratio;
4) and (3) drawing a calibration curve: performing regression analysis by using a least square method, and drawing a calibration curve;
5) detection of bile acids in samples: and (3) carrying out sampling and analysis on the sample by adopting the steps 2) and 3) to obtain the ratio of the bile acid in the sample to the internal standard, substituting the ratio into the calibration curve, and calculating the type and the content of the bile acid in the sample.
2. The method for detecting bile acid according to claim 1, wherein the conditions of the ultra high performance liquid chromatography in the step 2) are as follows: the chromatographic specification of C18 is 150 × 2.1mm,1.7 μm, liquid chromatogram A phase is 0.01% formic acid water solution, phase B is acetonitrile, column incubator temperature is 40 deg.C, sample tray is set at 4 deg.C, and injection volume is 3 μ L.
3. The method for detecting bile acid according to claim 1, wherein the conditions for mass spectrometry in step 3) are: spray voltage +3500/-3100V, shear gas (N2) flow rate 40, Aux gas (N2) flow rate 15, Sweep gas (N2) flow rate 0, Aux gas (N2) temperature 350 ℃, and campallarytemperture 320 ℃.
4. The method for detecting bile acid according to claim 1, wherein the parameters of the detection limit and the quantification limit of the method in step 3) are set as follows: the method minimum detection limit is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 3, and the method minimum quantitation limit is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 10.
5. The method for detecting bile acid according to claim 1, wherein the parameters for plotting the calibration curve in step 4) are set as: and (3) performing regression analysis by using a least square method, wherein when the weight is set to be 1/x, the recovery rate and the correlation coefficient of the calibration solution are best, and if the signal-to-noise ratio (S/N) of a certain calibration concentration is close to or less than 20 or the recovery rate exceeds the range of 80-120%, the concentration calibration point is excluded.
6. The method of detecting a bile acid of claim 1, further comprising: detecting the precision and accuracy of the method, wherein the precision of the method is evaluated by the standard relative deviation of the repeated sample injection of the QC sample; the accuracy is evaluated by the standard recovery rate of the QC sample, and the percentage value of the measured concentration and the standard concentration is the standard recovery rate.
7. The method of detecting a bile acid of claim 1, wherein said sample comprises serum, plasma, stool, liver tissue, intestinal contents, bile, gastric juice.
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Cited By (5)
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| CN111474256A (en) * | 2020-04-16 | 2020-07-31 | 上海中科新生命生物科技有限公司 | HP L C-MSMS-based quantitative analysis method for bile acid in feces |
| CN111830161A (en) * | 2020-07-21 | 2020-10-27 | 南京品生医学检验实验室有限公司 | Method for detecting 15 bile acids in serum |
| CN112505187A (en) * | 2020-12-11 | 2021-03-16 | 北京诺禾致源科技股份有限公司 | Method for detecting bile acid in excrement by UPLC-MS/MS (ultra Performance liquid chromatography-Mass Spectrometry/Mass Spectrometry) combination |
| CN114235995A (en) * | 2021-12-03 | 2022-03-25 | 天津国科医工科技发展有限公司 | Method for detecting 15 kinds of bile acids in serum |
| CN114509513A (en) * | 2021-12-29 | 2022-05-17 | 中国农业科学院饲料研究所 | Liquid chromatography high-resolution mass spectrometry qualitative and quantitative detection method for bile acid in multiple tissues |
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Cited By (5)
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| CN111474256A (en) * | 2020-04-16 | 2020-07-31 | 上海中科新生命生物科技有限公司 | HP L C-MSMS-based quantitative analysis method for bile acid in feces |
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| CN112505187A (en) * | 2020-12-11 | 2021-03-16 | 北京诺禾致源科技股份有限公司 | Method for detecting bile acid in excrement by UPLC-MS/MS (ultra Performance liquid chromatography-Mass Spectrometry/Mass Spectrometry) combination |
| CN114235995A (en) * | 2021-12-03 | 2022-03-25 | 天津国科医工科技发展有限公司 | Method for detecting 15 kinds of bile acids in serum |
| CN114509513A (en) * | 2021-12-29 | 2022-05-17 | 中国农业科学院饲料研究所 | Liquid chromatography high-resolution mass spectrometry qualitative and quantitative detection method for bile acid in multiple tissues |
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Application publication date: 20191220 |