CN111474288A - Mass spectrum kit for accurately measuring concentration of bile acid in serum - Google Patents
Mass spectrum kit for accurately measuring concentration of bile acid in serum Download PDFInfo
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Abstract
A mass spectrum kit for accurately determining the concentration of bile acid in serum, which is characterized in that: comprises a calibrator containing 15 bile acids, internal standards of deoxycholic acid-d4, glycodeoxycholic acid-d4 and taurodeoxycholic acid-d4, quality control products of the 15 bile acids, an extracting solution, a mobile phase and a complex solution. The method has the advantages that the complicated purification steps are not required, the required sample amount is small, and the method can be matched with a liquid chromatograph tandem mass spectrometer for use and applied to the inspection of clinical samples; meanwhile, the product can simultaneously detect 15 kinds of bile acids with different structures, achieves the function of single-sample multi-index synchronous detection, and has the characteristics of high accuracy, short detection time, less reagent consumption, convenient operation and the like; in addition, the measurement result can be directly traced to the reference system, thereby ensuring the accuracy of the measurement result.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a mass spectrum kit for accurately measuring the concentration of bile acid in serum.
Background
Bile Acids (BA) are the main components of bile, are synthesized by the liver, are the main metabolic pathways of endogenous cholesterol, and are also essential substances for digestion and absorption of lipid substances, and are classified into free bile acids including Cholic Acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), deoxycholic acid (UDCA) and lithocholic acid (1ithocholic acid, L CA) according to the structure, conjugated bile acids, which are mainly products of conjugated free bile acids with glycine or taurine, and are classified into primary bile acids (synthesized in hepatocytes) and secondary bile acids (synthesized in the intestinal tract), and the metabolic disorders of human bile acids and diseases of the hepatobiliary system and other diseases that can cause the metabolic disorders of bile acids, such as atherosclerosis, hypertension, etc., and the detection of the body components and the body contents of bile acids has important significance for the clinical understanding of the bile acid metabolism.
The toxicity of BA is related to its biochemical characteristics such as critical particle concentration and hydrophobicity, the toxicity of monohydroxy BA is the greatest, the toxicity of dihydroxy BA is the lowest, and the toxicity of trihydroxy BA is the least, each BA can individually act as a ligand to activate its corresponding receptor, which includes TGR5, FXR, PXR, PPAR α, CAR, VDR, etc.
The detection method mainly comprises a cyclic enzyme method, a high performance liquid chromatography (HP L C), capillary zone electrophoresis, electric chromatography, Gas Chromatography (GC), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (L C-MS/MS), the gas chromatography and the GC-MS have high selectivity and resolution when BA isomers are analyzed qualitatively and quantitatively, however, the technology is limited by a plurality of methods, mainly complex sample preparation and derivatization, particularly hydrolysis of a bound BA into a unbound form for analysis, the liquid chromatography resolution is not as good as GC, all BA isomers can not be separated, the high performance liquid chromatography (HP L C) method is still applicable to a wider high performance liquid tandem ultraviolet detection (HP L C-UV) method, the high performance liquid chromatography (HP L C) method is often used for high sensitivity, the high sensitivity of a high performance liquid tandem ultraviolet detection (HP L C-UV) method is often used for separation of all BA isomers, the high performance liquid chromatography-ultraviolet detection sensitivity is not good than a traditional high-specificity detection method, the high-specificity detection kit is also used for high-specificity detection of a high-specificity detection reagent kit, the detection kit is not suitable for detection of a high-specificity detection reagent, the detection kit is developed in a high-specificity detection kit, the detection kit is not suitable for a high-specificity detection kit, and a high-specificity detection kit is not suitable for detection kit, and is developed for a high-specificity detection kit, the detection kit is not suitable for a high-specificity detection kit, and is not suitable for detection kit, and is developed for detection of a high-specificity detection kit, and is developed for detection of a detection kit, and is developed for rapid detection of a detection kit, and is developed for detection of a high-specificity detection of a detection kit, and a detection of a high.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method which does not need to carry out complicated purification steps, has small required sample amount, can be matched with a liquid chromatograph tandem mass spectrometer for use and is applied to the inspection of clinical samples; meanwhile, the product can simultaneously detect 15 kinds of bile acids with different structures, achieves the function of single-sample multi-index synchronous detection, and has the characteristics of high accuracy, short detection time, less reagent consumption, convenient operation and the like; in addition, the measurement result can be directly traced to a reference system, so that the accuracy of the measurement result is ensured, and the mass spectrum kit is used for accurately measuring the concentration of the bile acid in the serum.
In order to solve the technical problems, the invention adopts the technical scheme that: a mass spectrometry kit for accurately determining the concentration of bile acids in human serum, the kit comprising: a calibrator comprising 15 bile acids; internal standards for deoxycholic acid-d4 (deoxycholic acid-2, 2,4,4-d4), glycodeoxycholic acid-d 4(CAS:1069132-37-1), and Taurodeoxycholic acid-d4 (taurodeoxocholic acid-d 4); 15 quality control products of bile acids; extracting solution; a mobile phase; and (5) compounding the solution.
The 15 kinds of bile acids are cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, and taurocholic acid.
The 15 bile acids in the calibrator have a series of concentration gradients respectively so as to complete the calibration.
Preferably, the concentration of each of the 15 bile acids in the calibrator ranges from 10 to 5000 nmol/L.
More preferably, the 15 bile acids in the calibrator respectively have a series of concentration gradients, and specifically, the calibrator containing six concentration gradients may be specifically, wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid are the same, and are sequentially 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L, 5000 nmol/L, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, and taurocholic acid are the same, and are sequentially 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L, and 1000 nmol/L.
The calibrators of the concentration gradients are all mixtures of 15 bile acids, for example, mixtures of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid and glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurocholic acid of a second concentration point of 100 nmol/L are used as calibrators of one gradient concentration, and the concentration gradients are in an appropriate range in clinical practical use, have good sensitivity and can be accurately measured by a detection instrument.
The internal standard is deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The concentration ranges of the three substances in the internal standard solution are all 100-500 nmol/L, the isotope markers are used as the internal standards, the concentrations of the isotope markers in the internal standard solution are limited, the sensitivity of internal standard substance detection can be guaranteed, and the effect of assisting in judging whether the efficiency of the pretreatment process and the detection conditions of instruments and the like are normal is achieved.
The quality control product comprises a quality control product I and a quality control product II, wherein the quality control product I comprises 15 bile acids with the concentration range of 50-250 nmol/L, the quality control product II comprises 15 bile acids with the concentration range of 300-600 nmol/L, the quality control product I and the quality control product II are both mixtures of 15 bile acids, the concentration range is the possible concentration range of each bile acid, namely the concentration of each bile acid component is the concentration in each mixture, two different concentration ranges of the quality control product I and the quality control product II are selected, each substance to be measured has stable property in the two concentration ranges, and the measurement precision is good.
Extracting solution: mixed solution of normal hexane and ethyl acetate, wherein the volume percentage of the ethyl acetate is 20-100%; the extracting solution is matched with methanol in the internal standard, so that protein in a serum sample can be removed through precipitation, and a substance to be detected is extracted into the supernatant, and the detection of the substance to be detected is not interfered.
Preferably, the volume percentage of ethyl acetate in the extract is 100%.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, and the mobile phase A and the mobile phase B both contain the same additional reagent, and the additional reagent comprises 2-10 mmol/L of ammonium acetate or ammonium formate and 0.05-0.20% of formic acid or acetic acid in percentage by volume.
Preferably, the external reagent in the mobile phase is preferably added with ammonium acetate and formic acid, wherein the concentration of the ammonium acetate is preferably 5 mmol/L, and the volume percentage of the formic acid is preferably 0.1%.
Compounding the solution: 50-100% methanol water solution; the sample can be well redissolved in the redissolution with the concentration, so that the 15 kinds of bile acids can be successfully and accurately detected in the detection process.
Preferably, the complex solution is a 65% methanol aqueous solution by volume.
The calibrator and the quality control material are mixture of 15 kinds of bile acid, and the mixture may be serum or lyophilized serum powder, methanol solution or lyophilized solution powder.
The invention also aims to provide a method for detecting the concentration of 15 bile acids in human serum by using the kit, and particularly a method for detecting by adopting liquid chromatography-tandem mass spectrometry (L C/MS/MS), which comprises the following steps:
(1) sample pretreatment:
① mixing the sample with the internal standard;
② adding extractive solution into the mixture obtained in step ①, and mixing;
③ centrifuging the mixed solution obtained in step ②, drying the supernatant, adding the redissolution to redissolve, mixing and centrifuging, and taking the supernatant for detection;
(2) carrying out L C/MS/MS detection analysis on the supernatant obtained after the redissolution in the step ③, and simultaneously detecting the supernatant and also comprising a calibrator and a quality control material;
(3) and (3) calculation of detection results:
a, drawing and fitting a calibration curve according to the detection and analysis result of the calibrator in the step (2);
b, calculating the recovery rate according to the detection and analysis result of the quality control product in the step (2);
and c, calculating the concentration of the analyte to be detected (namely the concentration of each 15 bile acid) according to the detection and analysis result of the sample supernatant obtained in the step (2) and the standard curve obtained in the step a.
In the step (1), the volume of the internal standard is equal to the volume of the sample, the volume of the extracting solution is 4-10 times of the volume of the sample, and vortex mixing is adopted for mixing.
The chromatographic conditions in the step (2) are that a chromatographic column is a C18 column or equivalent, mobile phases are a mobile phase A and a mobile phase B, the flow rate is 0.3m L/min, the gradient elution is finished by detecting the volume concentration change of the mobile phase B in terms of 0 min 65%, 1.5 min 78%, 5.2 min 78%, 5.21 min 100%, 7.5 min 100%, 7.51 min 65% and 10.5 min, the column temperature is 40 ℃, the sample injector temperature is 8 ℃, and the sample injection amount is 8 mu L;
the mass spectrum conditions are as follows: electrospray ion source, negative ion MRM scanning analysis, Q1/Q3 ion channel selected as cholic acid: 407.212 → 407.212, deoxycholic acid: 391.162 → 391.162, chenodeoxycholic acid: 391.125 → 391.125, ursodeoxycholic acid: 391.175 → 391.175, lithocholic acid: 375.162 → 375.162, deoxycholic acid-d 4: 395.162 → 395.162; glycocholic acid: 464.212 → 464.212, glycodeoxycholic acid: 448.212 → 448.212, glycochenodeoxycholic acid: 448.25 → 448.25, glycoursodeoxycholic acid: 448.162 → 448.162, glycolithocholic acid: 432.212 → 432.212, glycodeoxycholic acid-d 4: 452.212 → 452.212; taurocholic acid: 514.25 → 514.25, taurodeoxycholic acid: 498.212 → 498.212, taurochenodeoxycholic acid: 498.212 → 498.212, tauroursodeoxycholic acid: 498.212 → 498.212, taurocholic acid: 482.212 → 482.212, taurodeoxycholic acid-d 4: 502.212 → 502.212. Ion source parameters include spray voltage: 3000V, evaporation temperature: 300 ℃, ion transfer tube temperature: 200 ℃, sheath gas pressure: 50Arb, assist gas pressure: 10Arb, collision air pressure: 1 mToor.
The vortex mixing speed was 2000 rpm.
Adding the extracting solution in the step (1) into the sample, wherein the volume of the extracting solution is 8 times of the volume of the sample.
The ③ in the step (1) is centrifuged at low temperature, namely 4000rpm at 8 ℃, and the compound solution is added to be mixed and centrifuged at high speed at low temperature, namely 15000rpm at 8 ℃.
The drying process in ④ of the step (1) is nitrogen drying at 45 ℃.
The invention has the advantages and beneficial effects that:
1. the kit is used for treating a serum sample, and a very clean treatment solution can be obtained without complicated purification steps by a simple liquid-liquid extraction pretreatment method, and the required sample amount is small, so that the kit can be matched with a liquid chromatograph tandem mass spectrometer for use and applied to the inspection of clinical samples; meanwhile, the kit can detect 15 different bile acids simultaneously, achieves the function of single-sample multi-index synchronous detection, has the advantages of good detection specificity and high sensitivity, and has short time, high flux and low cost of the whole detection process.
2. The measurement result of the mass spectrometry kit method can be directly traced to a reference system, so that the accuracy of the measurement result is ensured.
3. The invention provides a kit for determining 15 kinds of bile acid concentration in human serum, which needs to accurately determine 15 kinds of bile acid simultaneously, and has the difficulty that firstly, a sample is a human serum sample, the interference of various other substances exists in the sample, and the sample treated by a reagent in the kit needs to be capable of eliminating the interference in the detection process, so that the substances to be detected are accurately stripped; secondly, 15 kinds of bile acid in human serum can be processed simultaneously, and a large amount of labor is consumed in determining and proportioning reagents; finally, the detection instrument is required to be capable of accurately and sensitively detecting the substance to be detected, the matching of the reagent and the detection instrument is required to be considered, the whole detection step is formulated, the operation parameters are determined, the chromatographic mass spectrometry condition is controlled, and the like; the above-mentioned technical problems are sufficiently solved by setting various specific components and specific concentrations in the kit of the present application.
Drawings
FIG. 1 shows 5 free bile acids (CA, DCA, CDCA, UDCA, L CA) and an internal standard (DCA-d)4) The corresponding mass chromatogram.
FIG. 2 shows 5 tauro-conjugated bile acids (TCA, TDCA, TCDCA, TUDCA, T L CA) and internal standard (TDCA-d)4) The corresponding mass chromatogram.
FIG. 3 shows 5 glycine-binding bile acids (GCA, GDCA, GCDCA, GUDCA, G L CA) and an internal standard (GDCA-d)4) The corresponding mass chromatogram.
FIG. 4 shows the standard curve corresponding to CA, the linear range is (10-5000) nmol/L.
FIG. 5 shows a standard curve corresponding to DCA, the linear range is (10-5000) nmol/L.
FIG. 6 shows a standard curve corresponding to CDCA, the linear range is (10-5000) nmol/L.
FIG. 7 shows a standard curve corresponding to UDCA, the linear range is (10-5000) nmol/L.
FIG. 8 shows a standard curve corresponding to L CA, the linear range is (10-5000) nmol/L.
FIG. 9 shows a standard curve corresponding to GCA, the linear range is (10-5000) nmol/L.
FIG. 10 shows the standard curve corresponding to GDCA, the linear range is (10-5000) nmol/L.
FIG. 11 shows the standard curve corresponding to GCDCA, the linear range is (10-5000) nmol/L.
FIG. 12 shows the standard curve corresponding to GUDCA, the linear range is (10-1000) nmol/L.
FIG. 13 shows a standard curve corresponding to G L CA, the linear range is (10-1000) nmol/L.
FIG. 14 shows the standard curve corresponding to TCA, the linear range is (10-1000) nmol/L.
FIG. 15 shows a standard curve corresponding to TDCA, the linear range is (10-1000) nmol/L.
FIG. 16 shows the standard curve corresponding to TCDCA, the linear range is (10-1000) nmol/L.
FIG. 17 shows the standard curve corresponding to TUDCA, with linear ranges of (10-1000) nmol/L.
FIG. 18 shows the standard curve corresponding to T L CA, the linear range is (10-1000) nmol/L.
Detailed Description
The present invention will be further described with reference to the following embodiments.
The invention provides a kit for determining 15 kinds of bile acid concentration in human serum, which needs to accurately determine 15 kinds of bile acid simultaneously, and has the difficulty that firstly, a sample is a human serum sample, the interference of various other substances exists in the sample, and the sample treated by a reagent in the kit needs to be capable of eliminating the interference in the detection process, so that the substances to be detected are accurately stripped; secondly, 15 kinds of bile acid in human serum can be processed simultaneously, and a large amount of labor is consumed in determining and proportioning reagents; finally, the detection instrument is required to be capable of accurately and sensitively detecting the substance to be detected, and the cooperation of the reagent and the detection instrument, the formulation of the whole detection step and the determination of each operation parameter are required to be considered.
In order to solve the above problems, the kit of the present invention is provided:
the calibrator comprises six concentration gradients, wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid and glycochenodeoxycholic acid are the same, and the six concentration points sequentially comprise 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L and 5000 nmol/L, and the six concentration points of glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurochenocholic acid are the same, and the six concentration points sequentially comprise 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L and 1000 nmol/L;
the calibrator with each concentration gradient is a mixture of 15 bile acids, for example, a mixture of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid and glycochenodeoxycholic acid of 50 nmol/L, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurocholic acid with the second concentration point of 100 nmol/L is taken as a calibrator with one gradient concentration, and the adopted series of concentration gradients are in a proper range in clinical practical use and have good sensitivity and can be accurately measured by a detection instrument;
internal standard: the internal standard solution is a solution containing deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The concentration ranges of the three substances in the internal standard solution are all 100-500 nmol/L;
the isotope label is used as an internal standard, and the concentration of the isotope label in an internal standard solution is limited, so that the sensitivity of internal standard substance detection can be ensured, and the effect of assisting in judging whether the efficiency of a pretreatment process and detection conditions such as instruments are normal or not is achieved;
quality control product: comprises a quality control product I and a quality control product II, wherein
The quality control product I comprises 15 kinds of bile acid with concentration range of 50-250 nmol/L;
the quality control product II comprises 15 kinds of bile acid with concentration range of 300-600 nmol/L;
the quality control product I and the quality control product II are both a mixture of 15 kinds of bile acid; the concentrations of the individual components are those in the respective mixtures; selecting two different concentration ranges of a quality control product I and a quality control product II, wherein each substance to be measured has stable property in the two concentration ranges and good measurement precision;
extracting solution: mixed solution of normal hexane and ethyl acetate, wherein the volume percentage of the ethyl acetate is 20-100%; the extracting solution is matched with methanol in the internal standard, so that protein in a serum sample can be removed through precipitation, and a substance to be detected is extracted into supernatant liquid, and the detection of the substance to be detected is not interfered;
the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, the mobile phase A and the mobile phase B both contain the same additional reagent, and the additional reagent is 2-10 mmol/L of ammonium acetate or ammonium formate and 0.05-0.20% of formic acid or acetic acid in volume percentage;
compounding the solution: 50-100% methanol water solution; the sample can be well redissolved in the redissolution with the concentration, so that the 15 kinds of bile acids can be successfully and accurately detected in the detection process.
In another improved embodiment, the calibrator and the quality control material are each a mixture of 15 kinds of bile acids, and the mixture can be serum or lyophilized serum powder, methanol solution or lyophilized solution powder.
In another modified embodiment, the volume percentage of ethyl acetate in the above-mentioned extract is preferably 100%.
In another modified embodiment, ammonium acetate and formic acid are preferably added to the mobile phase, wherein the concentration of ammonium acetate is preferably 5 mmol/L, and the volume percentage of formic acid is preferably 0.1%.
In another modified embodiment, the above-mentioned double solution is preferably a 65% methanol aqueous solution by volume.
Further, the kit is used for detecting by adopting liquid chromatography tandem mass spectrometry (L C/MS/MS), and the steps are as follows:
(1) sample processing
Taking a 100 mu L serum sample into a centrifugal tube of 5m L, adding an internal standard which is equal to the volume of the sample, namely 100 mu L internal standard, according to the concentration of each substance in the internal standard solution and the sensitivity of an instrument, carrying out vortex mixing for 1min, finally adding an extracting solution which is 4-10 times of the sample body, namely 400-1000 mu L extracting solution, carrying out vortex mixing for 5min, centrifuging for 5min, taking supernatant, carrying out nitrogen blow drying, adding 100 mu L redissolving solution, carrying out redissolving, carrying out high-speed centrifugation for 5min after uniform mixing, and taking 80 mu L supernatant for L C/MS/MS detection and analysis;
(2) l C/MS/MS detection analysis
(2.1) chromatographic conditions
Column chromatography: c18 column or equivalent;
the mobile phase: mobile phase a and mobile phase B;
flow rate 0.3m L/min;
gradient elution: as shown in table 1 below;
TABLE 1
In the gradient elution process in the table above, the volume ratio of the mobile phase a to the mobile phase B in the mobile phase is continuously adjusted and changed with time, and the change in the proportion of the mobile phase B is used as an indication.
Column temperature: 40 ℃;
sample injector temperature: 8 ℃;
sample size 8 μ L;
(2.2) Mass Spectrometry conditions
Electrospray ion Source (ESI), negative ion mode
Spray voltage: 3000V
Sheath gas pressure: 50Arb
Auxiliary gas pressure: 10Arb
The evaporation temperature: 350 deg.C
Temperature of ion transport tube: 200 deg.C
Collision air pressure: 1mToor
Multiple Reaction Monitoring (MRM) ion pairs, as shown in table 2 below:
TABLE 2
(2.3) injecting the processed calibrator, quality control product and sample into a liquid-quality continuous instrument with the diameter of 8 mu L respectively, and recording a chromatogram, wherein the processing method of the calibrator and the quality control product is the same as the sample processing process in the step (1) so as to ensure that the calibrator, the quality control product and the sample are processed equivalently and ensure that the detection result is reliable and accurate;
(3) calculation of detection results
(3.1) drawing and fitting of calibration curve: the general liquid chromatography tandem mass spectrometry system analysis software can automatically draw a calibration curve according to a calibration result, generally, the labeled concentration of 6 calibrators or the concentration ratio of the labeled concentration of the calibrators to an internal standard is used as a horizontal coordinate (x), the ratio of the actual measured peak area of the 6 calibrators to the peak area of each internal standard is used as a vertical coordinate (y), linear regression is performed by using a least square method, a standard curve is automatically drawn, and a regression equation can be obtained: and y is ax + b, wherein y is an ordinate, x is an abscissa, a is a slope, b is an intercept, and r (correlation coefficient) value is calculated, and is more than or equal to 0.9900.
And (3.2) calculating the recovery rate, wherein the measured concentration of each analyte of the quality control product can be obtained from a calibration curve according to the ratio of the peak areas of each analyte in the quality control product and the corresponding internal standard, the recovery rate is calculated according to the formula that the recovery rate (%) is measured concentration/marked concentration × 100, and the recovery rate (%) is within the range of 85-115%.
(3.3) calculation of sample results: the measured concentration of each analyte in the sample can be obtained from the calibration curve based on the ratio of the peak areas of each analyte in the sample and the corresponding internal standard.
In another improved embodiment, the vortex mixing speed in step (1) is 2000rpm (revolutions per minute), and at this speed, the mixed liquid is ensured to be completely mixed so that the substance to be detected can be completely extracted.
In another improved embodiment, the volume of the extracting solution added in the step (1) is 800 mu L, so that the substance to be tested can be completely extracted from the sample.
In another improved embodiment, the mixing and centrifugation of the extract added in the step (1) is low-temperature centrifugation, namely centrifugation at 4000rpm at 8 ℃; adding the complex solution, mixing and centrifuging to obtain low-temperature high-speed centrifugation, namely centrifuging at 15000rpm at 8 ℃. The centrifugal process is preferably performed at a low temperature to ensure the stability of the sample substance, and the low-speed and high-speed combined centrifugation can meet the aim of extracting the substance to be detected.
In another modified embodiment, the supernatant in the step (1) is dried by nitrogen at 45 ℃; and nitrogen is used as a protective atmosphere to ensure the stability of the substance to be detected in the sample.
Example 1
A kit for determining the concentration of 15 kinds of bile acids in human serum includes
The calibrator containing six concentration gradients is methanol solution freeze-dried powder of 15 bile acids;
wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid and glycochenodeoxycholic acid are the same, namely 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L and 5000 nmol/L, six concentration points of glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurochenolithocholic acid are the same, namely 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L and 1000 nmol/L;
internal standard: the internal standard solution is a solution containing deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The methanol solution of (1), wherein the concentration ranges of the three substances in the internal standard solution are all 250 nmol/L;
quality control product: comprises a quality control product I and a quality control product II which are serum freeze-dried powder of 15 bile acids; wherein
The concentration of 15 kinds of bile acid in the quality control product I is 250 nmol/L;
the concentration of 15 kinds of bile acid in the quality control product II is 500 nmol/L;
extracting solution: 100% ethyl acetate;
the mobile phase A is aqueous solution, the mobile phase B is methanol solution, and both the mobile phase A and the mobile phase B contain 5 mmol/L of ammonium acetate and 0.1% of formic acid by volume percentage;
compounding the solution: 65% methanol aqueous solution by volume.
The L C/MS/MS detection is carried out by using the kit, and the steps are as follows:
(1) test sample processing
Taking a 100 mu L serum sample to a centrifuge tube with the length of 5m L, adding a 100 mu L internal standard, carrying out vortex mixing at 2000rpm for 1min, finally adding 800 mu L extracting solution, carrying out vortex mixing at 2000rpm for 5min, carrying out centrifugation at 4000rpm at 8 ℃ for 5min, taking supernatant, carrying out nitrogen blow-drying at 45 ℃, adding 100 mu L redissolving solution, carrying out vortex mixing at 2000rpm for 1min, then carrying out centrifugation at 15000rpm for 5min at 8 ℃, and taking 80 mu L supernatant for L C/MS/MS detection and analysis;
(2) detection of sample L C/MS/MS detection analysis
(2.1) chromatographic conditions
Column chromatography: c18 column or equivalent;
the mobile phase: mobile phase a and mobile phase B;
flow rate 0.3m L/min;
gradient elution: as shown in Table 1;
column temperature: 40 ℃;
sample injector temperature: 8 ℃;
sample size 8 μ L;
(2.2) Mass Spectrometry conditions
Electrospray ion Source (ESI), negative ion mode
Spray voltage: 3000V
Sheath gas pressure: 50Arb
Auxiliary gas pressure: 10Arb
The evaporation temperature: 350 deg.C
Temperature of ion transport tube: 200 deg.C
Collision air pressure: 1mToor
Selective reaction monitoring ion pairs: as shown in Table 2;
example 2
A kit for determining the concentration of 15 kinds of bile acids in human serum includes
The calibrator containing six concentration gradients is serum freeze-dried powder of 15 bile acids;
wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid and glycochenodeoxycholic acid are the same, namely 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L and 5000 nmol/L, six concentration points of glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurochenolithocholic acid are the same, namely 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L and 1000 nmol/L;
internal standard: the internal standard solution is a solution containing deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4Wherein the concentration ranges of the three substances in the internal standard solution are all 250nmol/L；
Quality control product: comprises a quality control product I and a quality control product II which are serum freeze-dried powder of 15 bile acids; wherein
The concentration of 15 kinds of bile acid in the quality control product I is 250 nmol/L;
the concentration of 15 kinds of bile acid in the quality control product II is 500 nmol/L;
extracting solution: a mixed solution of n-hexane and ethyl acetate, wherein the volume percentage of the ethyl acetate is 50%;
the mobile phase A is aqueous solution, the mobile phase B is methanol solution, and both the mobile phase A and the mobile phase B contain 5 mmol/L ammonium formate and 0.1% formic acid by volume percentage;
compounding the solution: 65% methanol aqueous solution by volume.
L C/MS/MS detection was carried out using the above kit in the same manner as in example 1.
Example 3
A kit for determining the concentration of 15 kinds of bile acids in human serum includes
The calibrator containing six concentration gradients is methanol solution freeze-dried powder of 15 bile acids;
wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid and glycochenodeoxycholic acid are the same, namely 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L and 5000 nmol/L, six concentration points of glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurochenolithocholic acid are the same, namely 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L and 1000 nmol/L;
internal standard: the internal standard solution is a solution containing deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The methanol solution of (1), wherein the concentration ranges of the three substances in the internal standard solution are all 250 nmol/L;
quality control product: comprises a quality control product I and a quality control product II which are serum freeze-dried powder of 15 bile acids; wherein
The concentration of 15 kinds of bile acid in the quality control product I is 250 nmol/L;
the concentration of 15 kinds of bile acid in the quality control product II is 500 nmol/L;
extracting solution: a mixed solution of n-hexane and ethyl acetate, wherein the volume percentage of the ethyl acetate is 20%;
the mobile phase A is aqueous solution, the mobile phase B is methanol solution, and both the mobile phase A and the mobile phase B contain 10 mmol/L of ammonium acetate and 0.1% of acetic acid by volume percentage;
compounding the solution: 100% methanol.
L C/MS/MS detection was performed using the kit, and the amount of the extract added during the sample treatment was changed to 400. mu. L, as in example 1.
Example 4
A kit for determining the concentration of 15 kinds of bile acids in human serum includes
The calibrator with six concentration gradients is a methanol solution of 15 bile acids;
wherein six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid and glycochenodeoxycholic acid are the same, namely 10 nmol/L, 100 nmol/L, 500 nmol/L0, 1000 nmol/L1, 2500 nmol/L and 5000 nmol/L, six concentration points of glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurochenolithocholic acid are the same, namely 10 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L and 1000 nmol/L;
internal standard: the internal standard solution is a solution containing deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The methanol solution of (1), wherein the concentration ranges of the three substances in the internal standard solution are all 250 nmol/L;
quality control product: comprises a quality control product I and a quality control product II which are serum freeze-dried powder of 15 bile acids; wherein
The concentration of 15 kinds of bile acid in the quality control product I is 250 nmol/L;
the concentration of 15 kinds of bile acid in the quality control product II is 500 nmol/L;
extracting solution: 100% ethyl acetate;
the mobile phase A is aqueous solution, the mobile phase B is methanol solution, and both the mobile phase A and the mobile phase B contain 2 mmol/L of ammonium acetate and 0.1% of formic acid by volume percentage;
compounding the solution: 65% methanol aqueous solution by volume.
L C/MS/MS detection was carried out using the above kit in the same manner as in example 1.
The treated calibrator, quality control and sample obtained in examples 1 to 4 were each injected in an amount of 8 μ L using a mass spectrometer, a chromatogram was recorded, and the measurement results were calculated according to the following procedure:
(1) drawing and fitting a calibration curve: the general liquid chromatography tandem mass spectrometry system analysis software can automatically draw a calibration curve according to a calibration result, generally, the labeled concentration of 6 calibrators or the concentration ratio of the labeled concentration of the calibrators to an internal standard is used as a horizontal coordinate (x), the ratio of the actual measured peak area of the 6 calibrators to the peak area of each internal standard is used as a vertical coordinate (y), linear regression is performed by using a least square method, a standard curve is automatically drawn, and a regression equation can be obtained: and y is ax + b, wherein y is an ordinate, x is an abscissa, a is a slope, b is an intercept, and r (correlation coefficient) value is calculated, and is more than or equal to 0.9900.
(2) And (3) calculating the recovery rate, namely the measured concentration of each analyte of the quality control product can be obtained from a calibration curve according to the ratio of the peak areas of each analyte in the quality control product and the corresponding internal standard, wherein the recovery rate is calculated according to the formula that the recovery rate (%) is measured concentration/marked concentration × 100, and the recovery rate (%) is within the range of 85% -115%.
(3) Calculation of sample results: the measured concentration of each analyte in the sample can be obtained from the calibration curve based on the ratio of the peak areas of each analyte in the sample and the corresponding internal standard.
Fig. 1-3 are mass chromatograms corresponding to 15 bile acids and 3 internal standards, respectively.
The detection results of the embodiments 1-4 are basically consistent, and the analysis performance of the kit of the embodiment 1 is described by combining the related drawings as follows:
1. the standard curves and linearity are shown in FIGS. 4-11, the linear ranges of 8 analytes CA, DCA, CDCA, UDCA, L CA, GCA, GDCA and GCDCA are (10-5000) nmol/L, and the linear ranges of 7 analytes GUDCA, G L CA, TCA, TDCA, TCDCA, TUDCA and T L CA are (10-1000) nmol/L, and the correlation coefficients r of 15 bile acids are all greater than 0.99, with good linearity, as shown in FIGS. 12-18.
2. Accuracy (recovery experiment with mark)
The standard adding samples with two concentration gradients are respectively prepared, 3 parts of standard adding recovery is calculated after each concentration sample is repeatedly measured, and the results are shown in the following table 3.
TABLE 3
3. Precision degree
The results of the measurement of the quality control product I and the quality control product II are shown in Table 4 below, and CV values were all within 7%.
TABLE 4
According to the detection results, the kit and the liquid chromatography tandem mass spectrometry detection method have good accuracy, precision and linearity, can completely meet the requirements of clinical examination, and can simultaneously detect 15 different types of bile acids, thereby achieving the single-sample multi-index synchronous detection function.
Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the inspection and quarantine field if no special description is provided.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Claims (17)
1. A mass spectrum kit for accurately determining the concentration of bile acid in serum, which is characterized in that: comprises a calibrator containing 15 bile acids, internal standards of deoxycholic acid-d4, glycodeoxycholic acid-d4 and taurodeoxycholic acid-d4, quality control products of the 15 bile acids, an extracting solution, a mobile phase and a complex solution.
2. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein: the 15 kinds of bile acids are cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid and taurocholic acid; the 15 kinds of bile acids have a series of concentration gradients respectively.
3. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein the 15 bile acids respectively have a series of concentration gradients, specifically, six concentration points of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid in the calibrator are the same, namely 10 nmol/L, 100 nmol/L, 500 nmol/L, 1000 nmol/L, 2500 nmol/L, 5000 nmol/L, six concentration points of glycodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, 100 nmol/L, 250 nmol/L, 500 nmol/L, 1000 nmol/L are the same.
4. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein: the internal standard is deoxycholic acid-d4Glycodeoxycholic acid-d4And taurodeoxycholic acid-d4The concentration range of the three substances in the internal standard solution is 100-500 nmol/L.
5. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein: the quality control product comprises a quality control product I and a quality control product II,
the quality control product I comprises 15 kinds of bile acid with the concentration range of 50-250 nmol/L;
the quality control product II comprises 15 kinds of bile acid with the concentration range of 300-600 nmol/L.
6. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein: the extracting solution is a mixed solution of normal hexane and ethyl acetate, and the volume percentage of the ethyl acetate in the mixed solution is 20-100%.
7. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 6, wherein: the volume percentage of the ethyl acetate in the mixed solution was 100%.
8. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, and the mobile phase A and the mobile phase B both contain 2-10 mmol/L of ammonium acetate or ammonium formate and 0.05-0.20% by volume of formic acid or acetic acid, and the complex solution is 50-100% by volume of methanol aqueous solution.
9. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 8, wherein ammonium acetate and formic acid are added into the mobile phase A and the mobile phase B, the ammonium acetate concentration is 5 mmol/L, the formic acid volume percentage is 0.1%, and the complex solution is a 65% methanol aqueous solution by volume.
10. The mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 1, wherein: the calibrator and the quality control product are both a mixture of 15 kinds of bile acids, and the mixture is serum or serum freeze-dried powder, or methanol solution freeze-dried powder.
11. The method for detecting the mass spectrum kit for accurately determining the concentration of the bile acid in the serum according to any one of claims 1 to 10, which is characterized by comprising the following steps: the method comprises the following steps:
(1) sample pretreatment:
① mixing the sample with the internal standard;
② adding extractive solution into the mixture obtained in step ①, and mixing;
③ centrifuging the mixed solution obtained in step ②, drying the supernatant, adding the redissolution to redissolve, mixing and centrifuging, and taking the supernatant for detection;
(2) carrying out L C/MS/MS detection analysis on the supernatant obtained after the redissolution in the step ③, and simultaneously detecting the supernatant and also comprising a calibrator and a quality control material;
(3) and (3) calculation of detection results:
a, drawing and fitting a calibration curve according to the detection and analysis result of the calibrator in the step (2);
b, calculating the recovery rate according to the detection and analysis result of the quality control product in the step (2);
and c, calculating the concentration of the analyte to be detected according to the detection and analysis result of the sample supernatant obtained in the step (2) and the standard curve obtained in the step a.
12. The method for detecting mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 11, wherein the mass spectrometry kit comprises: in the step (1), the volume of the internal standard is equal to the volume of the sample, the volume of the extracting solution is 4-10 times of the volume of the sample, and vortex mixing is adopted for mixing.
13. The method for detecting mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 11, wherein the mass spectrometry kit comprises: in the step (2)
The chromatographic conditions comprise that a chromatographic column is a C18 column or equivalent, mobile phases are a mobile phase A and a mobile phase B, the flow rate is 0.3m L/min, the detection of gradient elution is finished by measuring the volume concentration change of the mobile phase B for 0 min 65%, 1.5 min 78%, 5.2 min 78%, 5.21 min 100%, 7.5 min 100%, 7.51 min 65% and 10.5 min, the column temperature is 40 ℃, the sample injector temperature is 8 ℃, and the sample injection amount is 8 mu L;
the mass spectrum conditions are as follows: electrospray ion source, negative ion MRM scanning analysis, Q1/Q3 ion channel selected as cholic acid: 407.212 → 407.212, deoxycholic acid: 391.162 → 391.162, chenodeoxycholic acid: 391.125 → 391.125, ursodeoxycholic acid: 391.175 → 391.175, lithocholic acid: 375.162 → 375.162, deoxycholic acid-d 4: 395.162 → 395.162; glycocholic acid: 464.212 → 464.212, glycodeoxycholic acid: 448.212 → 448.212, glycochenodeoxycholic acid: 448.25 → 448.25, glycoursodeoxycholic acid: 448.162 → 448.162, glycolithocholic acid: 432.212 → 432.212, glycodeoxycholic acid-d 4: 452.212 → 452.212; taurocholic acid: 514.25 → 514.25, taurodeoxycholic acid: 498.212 → 498.212, taurochenodeoxycholic acid: 498.212 → 498.212, tauroursodeoxycholic acid: 498.212 → 498.212, taurocholic acid: 482.212 → 482.212, taurodeoxycholic acid-d 4: 502.212 → 502.212. Ion source parameters include spray voltage: 3000V, evaporation temperature: 300 ℃, ion transfer tube temperature: 200 ℃, sheath gas pressure: 50Arb, assist gas pressure: 10Arb, collision air pressure: 1 mToor.
14. The method for detecting mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 11, wherein the mass spectrometry kit comprises: the vortex mixing speed was 2000 rpm.
15. The method for detecting mass spectrometry kit for accurately determining the concentration of bile acid in serum according to claim 11, wherein the mass spectrometry kit comprises: in the step (1), the volume of the extracting solution is 8 times of the sample volume.
16. The method for detecting the mass spectrum kit for accurately determining the concentration of bile acid in serum according to claim 11, wherein the centrifugation at ③ in the step (1) is low temperature centrifugation, i.e., 4000rpm centrifugation at 8 ℃, and the centrifugation with mixing of the double solutions is low temperature high speed centrifugation, i.e., 15000rpm centrifugation at 8 ℃.
17. The method for detecting the mass spectrum kit for accurately determining the concentration of the bile acid in the serum according to claim 11, wherein the drying process in ④ of the step (1) is nitrogen drying at 45 ℃.
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| CN112098532A (en) * | 2020-08-11 | 2020-12-18 | 杭州凯莱谱精准医疗检测技术有限公司 | Improved detection method for 15 bile acids in human serum by high performance liquid chromatography tandem mass spectrometry |
| CN112229937A (en) * | 2020-12-21 | 2021-01-15 | 北京大学第三医院（北京大学第三临床医学院） | Biomarkers and kits for diagnosis of polycystic ovarian syndrome and methods of use |
| CN113960222A (en) * | 2021-12-23 | 2022-01-21 | 中国中医科学院医学实验中心 | Serum sample-based bile acid full-channel metabolic profile detection method and application thereof |
| CN113960221A (en) * | 2021-12-23 | 2022-01-21 | 中国中医科学院医学实验中心 | Method for detecting bile acid full-channel metabolic profile based on fecal sample and application thereof |
| CN114894926A (en) * | 2022-04-27 | 2022-08-12 | 天津国科医工科技发展有限公司 | Bile acid detection method based on dry blood paper sheet method |
| CN116183780A (en) * | 2023-04-25 | 2023-05-30 | 天津云检医学检验所有限公司 | Absolute quantitative analysis method for bile acid in serum sample |
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| CN113960221A (en) * | 2021-12-23 | 2022-01-21 | 中国中医科学院医学实验中心 | Method for detecting bile acid full-channel metabolic profile based on fecal sample and application thereof |
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| CN113960222B (en) * | 2021-12-23 | 2022-02-25 | 中国中医科学院医学实验中心 | Serum sample-based bile acid full-channel metabolic profile detection method and application thereof |
| CN114894926A (en) * | 2022-04-27 | 2022-08-12 | 天津国科医工科技发展有限公司 | Bile acid detection method based on dry blood paper sheet method |
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Application publication date: 20200731 |