CN111665308A - Kit for detecting 15 bile acids in serum and application thereof - Google Patents
Kit for detecting 15 bile acids in serum and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting 15 bile acids in serum and application thereof, belonging to the technical field of blood detection. The 15 bile acids include: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid; the kit comprises: eluent, calibrator solution, mixed internal standard working solution, protein precipitator and quality control product. The kit is used for detecting the bile acid in the serum, a sample to be detected does not need derivatization treatment, the pretreatment is simple, the sample dosage is small, the sensitivity is high, the specificity is strong, the detection types are more, 15 kinds of bile acid can be simultaneously detected within 6.5 minutes, and the kit can be used for clinical diagnosis and health assessment of serum bile acid.
Description
Technical Field
The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting 15 bile acids in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology and application thereof.
Background
Bile acid is the main component in bile, is a generic name of 24-carbon cholanic acid hydroxyl derivatives, is the final product of cholesterol metabolism through liver tissues, and cannot be recycled when the liver circulation is destroyed. Bile acids can be classified into free bile acids and conjugated bile acids according to their structures. Free bile acids in the human body can be divided into primary and secondary bile acids according to their location and structure. Primary bile acid is converted from intrahepatic cholesterol, and Cholic Acid (CA) and Chenodeoxycholic acid (CDCA) are finally generated through a series of reactions. After the primary bile acid is synthesized in the liver cells, a series of transport proteins enter the capillary bile duct in the form of bile salt in the capillary bile duct membrane of the liver cells, the transport proteins are stored in a gall bladder, the gall bladder contracts after eating, the gall bladder is emptied, and the bile acid enters the intestinal tract. Under the action of bacteria in the intestinal tract, primary bile acids undergo hydrolysis, oxidation and epimerization with nuclear hydroxyl groups to produce bile acids, called secondary bile acids, including Deoxycholic acid (DCA), lithocholic acid (LCA) and Ursodeoxycholic acid (UDCA). Free bile acids are metabolites of cholesterol, and there are a number of complex mechanisms in the body that regulate the metabolism of free bile acids. Free bile acids play an important role in fat metabolism. Free bile acid exists mainly in liver and intestine circulation system, and can protect human body through recirculation. Bile acids are further classified into tauroconjugated bile acids and glycoconjugated bile acids. Taurocholic-bound bile acid refers to bile acid bound to taurine through an amide bond (peptide bond for short) to form Taurocholic-bound bile acid, which includes Taurocholic acid (TGA), taurochenodeoxycholic acid (TCDCA), Taurocholic acid (TLCA), Taurodeoxycholic acid (TDCA), and tauroursodeoxycholic acid (TUDCA). The glycine-binding bile acid refers to a bile acid which is bound to glycine by an amide bond (peptide bond for short) to form a glycine-binding bile acid, and includes Glycocholic acid (GCA), Glycochenodeoxycholic acid (GCDCA), glycolithocholic acid (GLCA), Glycodeoxycholic acid (GDCA), Glycoursodeoxycholic acid (GUDCA).
Serum bile acids can reflect anabolic uptake and excretion states of hepatocytes; the generation and metabolism of bile acid are closely related to liver, and when liver cells are diseased, the total bile acid in serum will change accordingly. Human endogenous bile acid is mainly hydrophobic bile acid, and when bile acid excretion is obstructed, the hydrophobic bile acid is highly compatible with fat, so that cell membrane and mitochondrial membrane structures are dissolved and damaged, cell apoptosis or death is caused, and liver injury is aggravated. However, the hydrophilic bile acid can promote the metabolism of endogenous bile acid, increase the contents of bile acid and phospholipid in bile, change the components of bile salt, reduce the toxicity of bile acid caused by hydrophobicity, and protect cell membrane and promote bile flow. However, the specificity of Total Bile Acid (TBA) in serum to different disease diagnoses is poor, and the detection result has very limited guiding effect on disease diagnosis and treatment. Total bile acid levels (TBA) have limited clinical significance except in some diseases such as pregnancy associated Intrahepatic Cholestasis (ICP). Therefore, the separation and the quantification of the multiple subtype bile acids can meet the requirements of clinical diagnosis and disease treatment.
In the prior art, methods for detecting various subtype bile acids based on LC-MS/MS are also available. CN 106841492A discloses a method for detecting five free bile acids in serum by high performance liquid chromatography tandem mass spectrometry, but the method has certain defects, such as only 5 free bile acids can be detected, the significance for disease diagnosis and clinical application is limited, after pretreatment protein precipitation, supernatant needs to be redissolved after freeze-drying by a centrifugal concentration freezing system, the pretreatment method is complex, the on-machine detection time reaches 12.5 minutes, and the method is difficult to apply in clinical detection. CN 107356694 a discloses a method for efficiently detecting 15 bile acids in blood, which can detect 15 bile acids simultaneously, but has certain defects, for example, the whole blood sample is dropped on a filter paper for pretreatment, and needs to be filtered and air-dried for more than 3 hours, the obtained dried blood is punched and sampled, and then extracted by liquid-liquid extraction and nitrogen-blowing concentration methods, and the method has the disadvantages of large sample amount, long pretreatment time, complex method, and limited clinical application. And the detection time on the machine reaches 10 minutes, so the application in clinical detection is difficult. CN 110596295 a discloses a method for detecting bile acid, which can detect fatty acid in serum, feces and liver samples though it detects more than 38 kinds of bile acid, but the serum and liver samples come from mouse and pelteobagrus fulvidraco, and the number of types detected in serum, feces and liver matrixes is different, and the biological reference interval of each bile acid in different matrixes is not specified, and the clinical reference meaning is unknown; the human body sample is a stool sample which is not as common as a blood sample and is not easy to obtain; the sample pretreatment method is complex and is limited in clinical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a kit for detecting 15 bile acids in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology and application thereof. The kit is used for 15 kinds of bile acid in blood samples, and is simple, efficient and reliable.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for detecting 15 kinds of bile acids in serum,
the 15 bile acids include: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid;
the kit comprises: eluent, calibrator solution, mixed internal standard working solution, protein precipitator and quality control product;
the eluent comprises an eluent A and an eluent B, wherein the eluent A is 0.01-0.1% formic acid-water solution, and the eluent B is methanol;
the calibrator solution comprises a mixed solution of cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid, which are prepared by a blank serum substrate and have known series concentrations;
the mixed internal standard working solution is a mixed solution of cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and taurodeoxycholic acid-d 4, which have known concentration and are prepared by methanol;
the quality control product comprises a mixed solution of cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid, which have known concentration and are prepared by a blank serum substrate.
Further, the eluent a was 0.01% formic acid-water solution.
Further, the protein precipitant is methanol.
Further, the blank serum matrix is 5% BSA aqueous solution. .
Further, the standard solution comprises mixed solutions with 7 concentration ratios.
Furthermore, the concentration of cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and tauroursodeoxycholic acid-d 4 in the mixed internal standard working solution is 50ng/mL cholic acid-d 4, 50ng/mL glycocholic acid-d 5, 5ng/mL taurocholic acid-d 5, 2.5ng/mL lithocholic acid-d 4, 2.5/mL glycolithocholic acid-d 4, 0.5ng/mL taurocholic acid-d 5, 50ng/mL deoxycholic acid-d 4, 25ng/mL glycodeoxycholic acid-d 5, 5ng/mL taurodeoxycholic acid-d 4, 75ng/mL chenodeoxycholic acid-d 4, 100ng/mL glycochenodeoxycholic acid-d 4, 25ng/mL taurodeoxycholic acid-d 5, 25ng/mL ursodeoxycholic acid-d 4, 25ng/mL glycoursodeoxycholic acid-d 4, and 1ng/mL taurodeoxycholic acid-d 4.
Further, the quality control product comprises mixed liquor with low, medium and high concentrations.
The kit is applied to detecting bile acid in serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology.
Has the advantages that: the kit is used for detecting the bile acid in serum, a sample to be detected does not need derivatization treatment, the pretreatment is simple, only one-step protein precipitation treatment is needed, the sample dosage is small, the sensitivity is high, the specificity is strong, the detection types are more, 15 kinds of bile acid can be simultaneously detected within 6.5 minutes, and the kit can be used for clinical diagnosis and health assessment of serum bile acid.
Drawings
FIG. 1 is a selective ion chromatogram of 15 bile acid standards of example 1.
FIG. 2 is a selective ion chromatogram of 15 bile acids in a serum sample of example 1.
Detailed Description
The invention provides a kit for detecting 15 kinds of bile acids in serum, which can be used for detecting the content of the bile acids in the serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology. The 15 bile acids include: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid.
The kit of the invention is used for separating a target object to be detected from interfering components in a serum matrix by using ultra-high performance liquid chromatography, detecting the mass-to-charge ratio (m/z) of the target object and a corresponding isotope internal standard thereof by using mass spectrometry, quantifying by using an isotope internal standard method, and respectively calculating the contents of 15 bile acids.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention respectively adopts cholic acid-d 4(CA-d4), glycocholic acid-d 5(GCA-d5), taurocholic acid-d 5(TCA-d5), lithocholic acid-d 4(LCA-d4), glycolithocholic acid-d 4(GLCA-d4), taurocholic acid-d 5(TLCA-d5), deoxycholic acid-d 4(DCA-d4), glycodeoxycholic acid-d 5(GDCA-d5), taurodeoxycholic acid-d 4(TDCA-d4), chenodeoxycholic acid-d 4(CDCA-d4), glycochenodeoxycholic acid-d 4(GCDCA-d4), taurodeoxycholic acid-d 5(TCDCA-d5), deoxycholic acid-d 4(UDCA-d4), glycoursodeoxycholic-d 4 (GUA-d 4) and taurocholic-d 4-4 as internal standard, the deuterated internal standard and the substance to be detected have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy in the determination of the bile acid in serum are better.
The kit comprises the following reagents:
(1) eluent:
eluent A: 0.01 to 0.1% formic acid-water solution; eluent B: methanol.
(2) Calibration solution:
preparing a mixed standard solution containing 20000ng/mL cholic acid, 20000ng/mL glycocholic acid, 2000ng/mL taurocholic acid, 1000ng/mL lithocholic acid, 1000ng/mL glycolithocholic acid, 200ng/mL taurocholic acid, 20000ng/mL deoxycholic acid, 10000ng/mL glycodeoxycholic acid, 2000ng/mL taurodeoxycholic acid, 30000ng/mL chenodeoxycholic acid, 40000ng/mL glycochenodeoxycholic acid, 10000ng/mL taurochenodeoxycholic acid, 10000ng/mL glycoursodeoxycholic acid, 400ng/mL taurodeoxycholic acid into calibration product solutions with blank serum matrixes at seven concentration points, wherein the seven concentration points of the calibration product solution are as follows:
the concentration of taurocholic acid is 0.08ng/mL, 0.2ng/mL, 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL in sequence;
the concentration of the tauroursodeoxycholic acid is 0.16ng/mL, 0.4ng/mL, 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL and 20ng/mL in sequence;
the concentrations of lithocholic acid and glycolithocholic acid are the same and are 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 25ng/mL and 50ng/mL in sequence;
the concentrations of cholic acid, glycocholic acid and deoxycholic acid are the same and are sequentially 8ng/mL, 20ng/mL, 40ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000 ng/mL;
the concentration of the taurocholic acid and the concentration of the taurodeoxycholic acid are the same, and the taurocholic acid and the taurodeoxycholic acid are 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in sequence;
the concentrations of glycodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid are the same and are 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL in sequence;
the concentration of the chenodeoxycholic acid is 12ng/mL, 30ng/mL, 60ng/mL, 150ng/mL, 300ng/mL, 750ng/mL and 1500ng/mL in sequence;
the concentration of glycochenodeoxycholic acid is 16ng/mL, 40ng/mL, 80ng/mL, 200ng/mL, 400ng/mL, 1000ng/mL and 2000ng/mL in sequence.
(3) Mixing internal standard working solution:
comprises 50ng/mL cholic acid-d 4, 50ng/mL glycocholic acid-d 5, 5ng/mL taurocholic acid-d 5, 2.5ng/mL lithocholic acid-d 4, 2.5ng/mL glycolithocholic acid-d 4, 0.5ng/mL taurocholic acid-d 5, 50ng/mL deoxycholic acid-d 4, 25ng/mL glycodeoxycholic acid-d 5, 5ng/mL taurodeoxycholic acid-d 4, 75ng/mL chenodeoxycholic acid-d 4, 100ng/mL glycodeoxycholic acid-d 4, 25ng/mL taurodeoxycholic acid-d 5, 25ng/mL deoxycholic acid-d 4, 25ng/mL glycoursodeoxycholic acid-d 4, 1ng/mL and taurodeoxycholic acid-d 4.
(4) Protein precipitant: methanol.
(5) Quality control products;
blank serum matrix solution containing 15 kinds of bile acids, divided into low, medium and high three concentrations, which are QC (L), QC (M) and QC (H), wherein:
QC (L) is that the mixed standard solution is diluted to 1000 times with blank serum matrix;
QC (M) is that the mixed standard solution is diluted to 200 times with blank serum matrix;
qc (h) diluted 20-fold with blank serum matrix for the mixed standard solution.
In a preferred embodiment, eluent a is 0.01% formic acid-water solution.
Bile acid is an endogenous substance, commercial bile acid-free blank serum cannot be obtained at present, the main component in human serum is serum protein, and bovine serum albumin is the most common human blank serum substitute matrix, so that Bovine Serum Albumin (BSA) is adopted as the human blank serum in the research, and when a standard solution is prepared, the blank serum matrix is 1-10% BSA aqueous solution; preferably 5% BSA in water.
The mixed standard solution mentioned in the present invention is prepared as follows:
(1) respectively adding pure methanol into cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid to be completely dissolved to prepare a standard substance mother liquor with the concentration of 5.00mg/mL, 4.00mg/mL, 5.00mg/mL, 2.00mg/mL, 5.00mg/mL and 10.00 mg/mL;
(2) and then pure methanol is used for preparing a mixed standard solution containing 20000ng/mL cholic acid, 20000ng/mL glycocholic acid, 2000ng/mL taurocholic acid, 1000ng/mL lithocholic acid, 1000ng/mL glycolithocholic acid, 200ng/mL taurocholic acid, 20000ng/mL deoxycholic acid, 10000ng/mL glycodeoxycholic acid, 2000ng/mL taurodeoxycholic acid, 30000ng/mL chenodeoxycholic acid, 40000ng/mL glycochenodeoxycholic acid, 10000ng/mL taurochenodeoxycholic acid, 10000ng/mL ursodeoxycholic acid, 10000ng/mL glycoursodeoxycholic acid and 400ng/mL taurodeoxycholic acid.
The mixed internal standard working solution provided by the invention is prepared according to the following method:
(1) weighing isotope internal standard substances, namely cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and taurodeoxycholic acid-d 4, respectively adding pure methanol to completely dissolve the substances, and preparing the substances into the isotope internal standard substances with the concentrations of 5mg/mL, 1mg/mL, 0.1mg/mL, 1mg/mL, 5mg/mL, 1mg/mL, 5mg/mL, and 1mg/mL of an isotopic internal standard mother liquor;
(2) the mother liquor of the isotope internal standard is prepared into a mother liquor containing 500ng/mL cholic acid-d 4, 500ng/mL glycocholic acid-d 5, 50ng/mL taurocholic acid-d 5, 25ng/mL lithocholic acid-d 4, 25ng/mL glycolithocholic acid-d 4, 5ng/mL taurocholic acid-d 5 and 500ng/mL deoxycholic acid-d 4 by pure methanol, isotopic internal standard SI solutions of 250ng/mL glycodeoxycholic acid-d 5, 50ng/mL taurodeoxycholic acid-d 4, 750ng/mL chenodeoxycholic acid-d 4, 1000ng/mL glycochenodeoxycholic acid-d 4, 250ng/mL taurochenodeoxycholic acid-d 5, 250ng/mL ursodeoxycholic acid-d 4, 250ng/mL glycoursodeoxycholic acid-d 4, and 10ng/mL and tauroursodeoxycholic acid-d 4;
(3) taking 100 mu L of SI solution, adding 900 mu L of methanol-water solution, and uniformly mixing to obtain mixed internal standard working solution;
when the mixed internal standard working solution is prepared, the methanol-water solution is 80-95% methanol-water solution; preferably an 80% methanol-water solution.
In a more preferred embodiment, the reagent kit for detecting 15 bile acids in serum by ultra performance liquid chromatography tandem mass spectrometry comprises the following reagents:
(1) eluent:
eluent A: 0.01% formic acid-water solution; eluent B: methanol.
(2) Calibration solution:
weighing each standard substance to be detected, including cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid, respectively adding pure methanol to completely dissolve, and preparing into standard substance mother liquor with the concentration of 5.00mg/mL, 5.00mg/mL and 10.00mg/mL in sequence;
preparing a mixed standard solution containing 20000ng/mL cholic acid, 20000ng/mL glycocholic acid, 2000ng/mL taurocholic acid, 1000ng/mL lithocholic acid, 1000ng/mL glycolithocholic acid, 200ng/mL taurocholic acid, 20000ng/mL deoxycholic acid, 10000ng/mL glycodeoxycholic acid, 2000ng/mL taurodeoxycholic acid, 30000ng/mL chenodeoxycholic acid, 40000ng/mL glycochenodeoxycholic acid, 10000ng/mL taurochenodeoxycholic acid, 10000ng/mL ursodeoxycholic acid, 10000ng/mL glycoursodeoxycholic acid and 400ng/mL taurodeoxycholic acid from the mother solution of each standard substance by using pure methanol;
the mixed standard solutions were formulated with 5% BSA to make up seven calibrator solutions at different concentration points.
(3) Mixing internal standard working solution:
weighing isotope internal standard substances, namely cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and taurodeoxycholic acid-d 4, respectively adding pure methanol to completely dissolve the substances, and preparing the substances into the isotope internal standard substances with the concentrations of 5mg/mL, 1mg/mL, 0.1mg/mL, 1mg/mL, 5mg/mL, 1mg/mL, 5mg/mL, and 1mg/mL of an isotopic internal standard mother liquor;
the mother liquor of the isotope internal standard is prepared into a mother liquor containing 500ng/mL cholic acid-d 4, 500ng/mL glycocholic acid-d 5, 50ng/mL taurocholic acid-d 5, 25ng/mL lithocholic acid-d 4, 25ng/mL glycolithocholic acid-d 4, 5ng/mL taurocholic acid-d 5 and 500ng/mL deoxycholic acid-d 4 by pure methanol, isotopic internal standard SI solutions of 250ng/mL glycodeoxycholic acid-d 5, 50ng/mL taurodeoxycholic acid-d 4, 750ng/mL chenodeoxycholic acid-d 4, 1000ng/mL glycochenodeoxycholic acid-d 4, 250ng/mL taurochenodeoxycholic acid-d 5, 250ng/mL ursodeoxycholic acid-d 4, 250ng/mL glycoursodeoxycholic acid-d 4, and 10ng/mL and tauroursodeoxycholic acid-d 4;
and adding 900 mu L of 80% methanol-water solution into 100 mu L of SI solution, and uniformly mixing to obtain the mixed internal standard working solution.
(4) Protein precipitant: methanol.
(5) Quality control product:
preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using 5% BSA; the concentrations of QC (L), QC (M), QC (H) for the 15 bile acids are shown in Table 1.
TABLE 1 corresponding concentration of quality control (unit ng/mL)
In a preferred embodiment, the calibrator solution is prepared as follows:
accurately weighing 3-5mg of each standard substance powder to be detected in a 5mL centrifuge tube (the standard substances with the specification below 3mg are not required to be weighed and are completely dissolved), preparing the mother solution concentration of the standard substance in the following table by using pure methanol, preparing the mixed standard solution (detailed in table 2) by using pure methanol at each use concentration, and uniformly mixing for later use.
TABLE 2 preparation of the Mixed Standard solutions
Adding 20 μ L of the mixed standard solution to 380 μ L of 5% BSA as a first high-value concentration point; diluting the first high-value concentration point with 5% BSA with the same volume to obtain a second high-value concentration point; diluting the first high-value concentration point with 4 times of volume of 5% BSA to obtain a third high-value concentration point; diluting the second high-value concentration point with 4 times of volume of 5% BSA to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 4 times of volume of 5% BSA to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 4 times of volume of 5% BSA to obtain a sixth high-value concentration point; the fifth high concentration point was diluted with 4 volumes of 5% BSA to obtain the seventh high concentration point.
The seven concentration points of the calibrator solution were:
the concentration of taurocholic acid is 0.08ng/mL, 0.2ng/mL, 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL in sequence;
the concentration of the tauroursodeoxycholic acid is 0.16ng/mL, 0.4ng/mL, 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL and 20ng/mL in sequence;
the concentrations of lithocholic acid and glycolithocholic acid are the same and are 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 25ng/mL and 50ng/mL in sequence;
the concentrations of cholic acid, glycocholic acid and deoxycholic acid are the same and are sequentially 8ng/mL, 20ng/mL, 40ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000 ng/mL;
the concentration of the taurocholic acid and the concentration of the taurodeoxycholic acid are the same, and the taurocholic acid and the taurodeoxycholic acid are 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in sequence;
the concentrations of glycodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid are the same and are 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL in sequence;
the concentration of the chenodeoxycholic acid is 12ng/mL, 30ng/mL, 60ng/mL, 150ng/mL, 300ng/mL, 750ng/mL and 1500ng/mL in sequence;
the concentration of glycochenodeoxycholic acid is 16ng/mL, 40ng/mL, 80ng/mL, 200ng/mL, 400ng/mL, 1000ng/mL and 2000ng/mL in sequence.
In a preferred embodiment, the mixed internal standard working solution is prepared according to the following method:
accurately weighing 3-5mg of each isotope internal standard substance into a 5mL centrifuge tube (standard substances with the specification below 3mg are not required to be weighed and are completely dissolved), preparing isotope internal standard mother liquor with pure methanol into the concentrations in the following table, preparing isotope mixed internal standard SI solution (detailed in table 3) with pure methanol solution with each use concentration, finally taking 100 mu L of SI solution, adding 900 mu L of 80% methanol-water solution, and uniformly mixing to obtain the mixed internal standard working solution.
TABLE 3 preparation of SI solution as isotopic mixing internal standard
The concentration of the methanol-water solution referred to in the present invention generally refers to the volume concentration.
The application of the kit in detecting 15 kinds of bile acids in serum by using the ultra performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting 15 bile acids in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology is characterized in that the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the bile acids in the pretreated serum, an object to be detected is firstly separated from interfering components in a serum matrix by using the ultra-high performance liquid chromatography, then the mass spectrum is used to detect the mass-to-charge ratio (m/z) of the object and a corresponding isotope internal standard thereof, the isotope internal standard method is used for quantification, the content of the 15 bile acids is respectively calculated, and the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.01 to 0.1% formic acid-water solution; mobile phase B: methanol;
the type of the chromatographic column: ACQUITY UPLC BEH C18(2.1×50mm,1.7μm)；
And (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 60:40 to 2:98 at a constant speed within 0-4.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 2:98 within 4.0-4.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is 40:60 within 4.5-6.5 minutes;
(2) mass spectrum conditions:
performing negative ion scanning in an electrospray ionization (ESI) mode by using multi-reaction monitoring (MRM); the spray voltage was 2.5kV (ESI-); the ion source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; 15 bile acids and their corresponding isotopic internal standards were monitored simultaneously.
In a preferred embodiment, the chromatographic column is a C18 column.
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid into the mobile phase A, can effectively improve the ionization efficiency of certain target compounds, has higher sensitivity in detecting bile acid by adopting an LC-MS/MS method in the prior art under the coordination of other conditions, has simple pretreatment, only needs one-step protein precipitation treatment, has small sample dosage and more detection types, and can simultaneously detect 15 bile acids within 6.5 minutes. Under the condition of not influencing the effect of the invention, in a preferable scheme, the mobile phase A is 0.01-0.1% formic acid-water solution. In a more preferred embodiment, mobile phase a is a 0.01% formic acid-water solution.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts 0.01-0.5% formic acid-water solution and methanol as mobile phase, and the type of chromatographic column is as follows: ACQUITYUPLC BEH C18(2.1 × 50mm,1.7 μm), the chromatographic column is C18 column, under the coordination of other conditions, the endogenous substance does not interfere the determination of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, the separation and the detection can be completed within 6.5min, and the precision and the accuracy meet the requirements.
In one embodiment, the flow rate is 0.3-0.5 mL/min, preferably 0.4 mL/min.
Further, the column temperature is 35-45 ℃, and preferably 40 ℃.
Furthermore, the injection volume is 0.5-5 μ L, preferably 1 μ L.
In a preferred scheme, when the ultra performance liquid chromatography tandem mass spectrometry technology is adopted to detect 15 kinds of bile acids in serum, the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.01% formic acid-water solution; mobile phase B: methanol;
the type of the chromatographic column: ACQUITY UPLC BEH C18(2.1×50mm,1.7μm)；
The gradient elution mode is adopted, and is shown in the table 4; the flow rate was 0.4mL/min, the column temperature was 40 ℃ and the injection volume was 1. mu.L.
TABLE 4 mobile phase gradient elution parameters
| Time of day | Flow rate (mL/min) | ％A | ％B | Curve |
| 0.0 | 0.4 | 60 | 40 | - |
| 4.0 | 0.4 | 2 | 98 | 6 |
| 4.5 | 0.4 | 2 | 98 | 6 |
| 6.5 | 0.4 | 60 | 40 | 1 |
(2) Mass spectrum conditions:
performing negative ion scanning in an electrospray ionization (ESI) mode by using multi-reaction monitoring (MRM); the spray voltage was 2.5kV (ESI-); the ion source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; 15 kinds of bile acids and corresponding isotope internal standards thereof are monitored simultaneously, and the mass spectrum acquisition parameters of each target object to be detected are shown in table 5.
TABLE 5 bile acid profile parameters
| Compound (I) | Parent ion (m/z) | Ionic acid (m/z) | Taper hole voltage (V) | Collision voltage (V) |
| CA | 407.3 | 407.3 | 40 | 4 |
| CA-d4 | 411.3 | 411.3 | 40 | 4 |
| GCA | 464.3 | 73.95 | 40 | 38 |
| GCA-d5 | 469.3 | 73.95 | 40 | 38 |
| TCA | 514.35 | 80.01 | 40 | 60 |
| TCA-d5 | 519.3 | 79.9 | 40 | 60 |
| LCA | 375.3 | 375.3 | 40 | 4 |
| LCA-d4 | 379.3 | 379.3 | 40 | 4 |
| GLCA | 432.3 | 73.95 | 40 | 34 |
| GLCA-d4 | 437.1 | 73.95 | 40 | 34 |
| TLCA | 482.3 | 80.01 | 40 | 54 |
| TLCA-d5 | 487.3 | 79.9 | 40 | 62 |
| DCA | 391.3 | 391.3 | 40 | 4 |
| DCA-d4 | 395.3 | 395.3 | 40 | 4 |
| GDCA | 448.3 | 73.95 | 40 | 34 |
| GDCA-d5 | 453.3 | 73.95 | 40 | 34 |
| TDCA | 498.3 | 80.1 | 40 | 55 |
| TDCA-d4 | 502.3 | 80.1 | 40 | 55 |
| CDCA | 391.3 | 391.3 | 40 | 4 |
| CDCA-d4 | 395.3 | 395.3 | 40 | 4 |
| GCDCA | 448.3 | 73.95 | 40 | 34 |
| GCDCA-d4 | 452.3 | 73.95 | 40 | 34 |
| TCDCA | 498.3 | 80.1 | 40 | 55 |
| TCDCA-d5 | 503.3 | 79.9 | 40 | 60 |
| UDCA | 391.3 | 391.3 | 40 | 4 |
| UDCA-d4 | 395.3 | 395.3 | 40 | 4 |
| GUDCA | 448.3 | 73.95 | 40 | 34 |
| GUDCA-d4 | 452.3 | 73.95 | 40 | 34 |
| TUDCA | 498.3 | 80.1 | 40 | 55 |
| CA | 407.3 | 407.3 | 40 | 4 |
In one protocol, pre-treated serum was prepared as follows: adding the mixed internal standard working solution into the serum, adding a protein precipitator after vortex, and taking supernatant after vortex centrifugation; wherein the protein precipitant is methanol.
In a preferred embodiment, the pre-treated serum is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard into the centrifuge tube for working, adding 180 mu L of methanol after vortex, oscillating for 4-10 min, then, 12000-15000 r/min, centrifuging for 4-10 min at 10-20 ℃, and taking 70 mu L of supernatant.
In a more preferred embodiment, the pre-treated serum is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard for working, and then whirling for 5 s; adding 180 μ L methanol, and oscillating at high speed (maximum vibration speed) for 5 min; centrifuging at 14000r/min at 15 ℃ for 5 min; 70 mu L of the supernatant was put into a sample injection bottle with an internal cannula and tested, and the sample injection amount was 1 mu L.
When a standard curve is drawn, 50 mu L of each calibrator concentration point sample is taken, 20 mu L of mixed internal standard is added to work, and then vortex is carried out for 5 s; adding 180 μ L methanol, and oscillating at high speed (maximum vibration speed) for 5 min; centrifuging at 14000r/min at 15 ℃ for 5 min; 70 mu L of the supernatant was put into a sample injection bottle with an internal cannula and tested, and the sample injection amount was 1 mu L.
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1
First, experimental material and instrument
1. Material
The samples for the study experiments in the kit were obtained from serum samples collected from the clinic in 2019 and 4 months in the heart disease hospital in Wuhan Asia.
(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.
(2) Reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); chromatography column model ACQUITY UPLC BEH C18(2.1 × 50mm,1.7 μm) (Waters, USA).
(3) And (3) standard substance: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid were purchased from carbofuran; glycolithocholic acid and taurocholic acid were purchased from TRC; cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, taurocholic acid-d 5, glycodeoxycholic acid-d 5, chenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, and ursodeoxycholic acid-d 4 were purchased from TRC; deoxycholic acid-d 4, glycolithocholic acid-d 4, taurodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4, tauroursodeoxycholic acid-d 4 were purchased from Sigma; glycchenodeoxycholic acid-d 4 was purchased from CIL.
(4) Quality control product: the blank serum matrix solution containing 15 kinds of bile acids has low, medium and high concentration, and is QC (L), QC (M) and QC (H), respectively.
The upper and lower peripheries of the kit are coated with films, the kit is shockproof and heat-insulated, mobile phases A and B are placed on the upper left, and 11 ampoules are respectively placed on the lower left, wherein the standard solution and the quality control product are respectively; 50mL of protein precipitant was placed on the right side.
Second, liquid condition
1. Chromatographic conditions are as follows: mobile phase A: 0.01% formic acid-water solution; mobile phase B: methanol. The type of the chromatographic column: ACQUITYUPLC BEH C18(2.1 × 50mm,1.7 μm), gradient elution was used, as detailed in Table 4, the flow rate was 0.4mL/min, the column temperature was 40 ℃ and the injection volume was 1 μ L.
2. Mass spectrum conditions: performing negative ion scanning in an electrospray ionization (ESI) mode by using multi-reaction monitoring (MRM); the spray voltage was 2.5kV (ESI-); the ion source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; 15 kinds of bile acids and corresponding isotope internal standards thereof are monitored simultaneously, and the mass spectrum acquisition parameters of each target object to be detected are shown in table 5.
Third, the experimental process
1. Standard preparation
Accurately weighing 3-5mg of each standard substance powder to be detected in a 5mL centrifuge tube (standard substances with the specification below 3mg are not required to be weighed and are completely dissolved), preparing the mother solution concentration of the standard substance in the following table by using pure methanol, preparing the mixed standard solution (detailed in table 2) by using the pure methanol solution with each use concentration, and uniformly mixing for later use.
The mixed standard solution is prepared into calibration solution of seven different concentration points by using a blank serum matrix (5% BSA), and the preparation process is as follows:
adding 20 μ L of the mixed standard solution to 380 μ L of 5% BSA as a first high-value concentration point; diluting the first high-value concentration point with 5% BSA with the same volume to obtain a second high-value concentration point; diluting the first high-value concentration point with 4 times of volume of 5% BSA to obtain a third high-value concentration point; diluting the second high-value concentration point with 4 times of volume of 5% BSA to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 4 times of volume of 5% BSA to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 4 times of volume of 5% BSA to obtain a sixth high-value concentration point; the fifth high concentration point was diluted with 4 volumes of 5% BSA to obtain the seventh high concentration point.
The seven concentration points of the calibrator solution were:
the concentration of taurocholic acid is 0.08ng/mL, 0.2ng/mL, 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL in sequence;
the concentration of the tauroursodeoxycholic acid is 0.16ng/mL, 0.4ng/mL, 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL and 20ng/mL in sequence;
the concentrations of lithocholic acid and glycolithocholic acid are the same and are 0.4ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 25ng/mL and 50ng/mL in sequence;
the concentrations of cholic acid, glycocholic acid and deoxycholic acid are the same and are sequentially 8ng/mL, 20ng/mL, 40ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000 ng/mL;
the concentration of the taurocholic acid and the concentration of the taurodeoxycholic acid are the same, and the taurocholic acid and the taurodeoxycholic acid are 0.8ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in sequence;
the concentrations of glycodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid are the same and are 4ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL in sequence;
the concentration of the chenodeoxycholic acid is 12ng/mL, 30ng/mL, 60ng/mL, 150ng/mL, 300ng/mL, 750ng/mL and 1500ng/mL in sequence;
the concentration of glycochenodeoxycholic acid is 16ng/mL, 40ng/mL, 80ng/mL, 200ng/mL, 400ng/mL, 1000ng/mL and 2000ng/mL in sequence.
2. Preparation of mixed internal standard working solution
Accurately weighing 3-5mg of each isotope internal standard substance into a 5mL centrifuge tube (standard substances with the specification below 3mg are not required to be weighed and are completely dissolved), preparing isotope internal standard mother liquor with pure methanol into the concentrations in the following table, preparing isotope mixed internal standard SI solution (detailed in table 3) with pure methanol solution with each use concentration, finally taking 100 mu L of SI solution, adding 900 mu L of 80% methanol-water solution, and uniformly mixing to obtain the mixed internal standard working solution.
3. Preparation of quality control product
The mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using 5% BSA.
QC (L) includes: 20ng/mL Cholic Acid (CA), 20ng/mL glycocholic acid (GCA), 2ng/mL taurocholic acid (TCA), 1ng/mL lithocholic acid (LCA), 1ng/mL glycolithocholic acid (GLCA), 0.2ng/mL taurocholic acid (TLCA), 20ng/mL deoxycholic acid (DCA), 10ng/mL glycodeoxycholic acid (GDCA), 2ng/mL taurodeoxycholic acid (TDCA), 30ng/mL chenodeoxycholic acid (CDCA), 40ng/mL glycochenodeoxycholic acid (GCDCA), 10ng/mL taurodeoxycholic acid (TCDCA), 10ng/mL ursodeoxycholic acid (UDCA), 10ng/mL glycoursodeoxycholic acid (GUDCA), 0.4ng/mL taurodeoxycholic acid (TUDCA).
QC (M) comprises: 100ng/mL Cholic Acid (CA), 100ng/mL glycocholic acid (GCA), 10ng/mL taurocholic acid (TCA), 5ng/mL lithocholic acid (LCA), 5ng/mL glycolithocholic acid (GLCA), 1ng/mL taurocholic acid (TLCA), 100ng/mL deoxycholic acid (DCA), 50ng/mL glycodeoxycholic acid (GDCA), 10ng/mL taurodeoxycholic acid (TDCA), 150ng/mL chenodeoxycholic acid (CDCA), 200ng/mL glycochenodeoxycholic acid (GCDCA), 50ng/mL taurodeoxycholic acid (TCDCA), 50ng/mL ursodeoxycholic acid (UDCA), 50ng/mL glycoursodeoxycholic acid (GUDCA), 2ng/mL taurodeoxycholic acid (TUDCA).
QC (H) includes: 400ng/mL Cholic Acid (CA), 400ng/mL glycocholic acid (GCA), 40ng/mL taurocholic acid (TCA), 20ng/mL lithocholic acid (LCA), 20ng/mL glycolithocholic acid (GLCA), 4ng/mL taurocholic acid (TLCA), 400ng/mL deoxycholic acid (DCA), 200ng/mL glycodeoxycholic acid (GDCA), 40ng/mL taurodeoxycholic acid (TDCA), 600ng/mL chenodeoxycholic acid (CDCA), 800ng/mL glycochenodeoxycholic acid (GCDCA), 200ng/mL taurodeoxycholic acid (TCDCA), 200ng/mL ursodeoxycholic acid (UDCA), 200ng/mL glycoursodeoxycholic acid (GUDCA), 8ng/mL taurodeoxycholic acid (TUDCA).
4. Sample processing
1) Pretreatment of a standard product: taking 50 mu L of sample at each concentration point, adding 20 mu L of mixed internal standard for working, and then whirling for 5 s; adding 180 μ L methanol, and oscillating at high speed (maximum vibration speed) for 5 min; centrifuging at 14000r/min at 15 ℃ for 5 min; 70 mu L of the supernatant was put into a sample injection bottle with an internal cannula and tested, and the sample injection amount was 1 mu L.
2) Pretreatment of a serum sample: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard for working, and then whirling for 5 s; adding 180 μ L methanol, and oscillating at high speed (maximum vibration speed) for 5 min; centrifuging at 14000r/min at 15 ℃ for 5 min; 70 mu L of the supernatant was put into a sample injection bottle with an internal cannula and tested, and the sample injection amount was 1 mu L.
3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.
The components of the assay kit are shown in Table 6.
TABLE 6 bile acid assay kit Components (100 parts)
Fourth, method verification
In the detection method of the invention, the peak shapes of the bile acid standard substance and the serum sample are symmetrical without interfering with other peaks, which indicates that good detection can be obtained under the condition, and fig. 1 is a selective ion flow chromatogram of the bile acid standard substance; FIG. 2 is a selective ion flow chromatogram of bile acids in a serum sample.
1. Standard curve:
and (3) establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the bile acid to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of 15 kinds of bile acid in the respective concentration range has good linearity and the correlation coefficient is above 0.99, which is detailed in table 7.
TABLE 715 retention times and Linear Range of bile acids
2. Minimum limit of quantitation
The lowest limit of quantitation (LLOQ), which is the lowest point of the standard curvilinear range, also reflects the sensitivity of the method. Part of bile acid has low content in human body, has higher requirements on sensitivity and specificity of the method, optimizes and investigates the method, the current minimum quantitative limit (LLOQ) basically meets the sensitivity requirement of simultaneous detection of 15 bile acids, and the concentration of the LLOQ is specifically shown in Table 8.
TABLE 8 quantitative lower limit data table
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. Randomly selecting a sample of human serum, wherein 1 sample is not added with the standard substance, and the other 3 samples are respectively added with the standard substances with low, medium and high concentrations, repeatedly treating and measuring for 5 times by the same steps, and calculating the recovery rate result, which is shown in table 9. The results show that the results of the standard recovery rate of 15 bile acids in the serum are between 85.72% and 114.54%, and the RSD of 5 times of repeated tests is in the range of 7.17% to 13.12%, and all the results meet the requirements.
TABLE 9 results of recovery of bile acids in serum normalized to ng/mL
4. And (3) precision test: taking normal human serum samples, repeatedly processing 6 batches within one day, processing for 3 days, quantitatively measuring the concentration of bile acid by using an isotope internal standard method, continuously carrying out statistics on the internal batch precision every day for three days, calculating the internal batch precision to be 0.8-15.15%, carrying out processing for 3 batches within three days, calculating the inter-batch precision to be 2.94-13.70%, and obtaining the results of the inter-batch precision shown in Table 10.
TABLE 10 results of precision test within and between batches (unit ng/mL)
From the results, the kit can measure 15 bile acids in human serum by an UPLC-MS/MS method, and simultaneously detects the peak time and ion pair of the target object, so that the kit has high sensitivity and strong specificity. Meanwhile, the isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the influence of the conditions such as a pretreatment process, a sample loading volume and flow is avoided, and accurate quantification can be achieved.
The standard recovery rates of 15 bile acids in serum are considered to be 85-115%, and the recovery rates meet the requirements.
The reproducibility results of the method show that the intra-day precision, the inter-day precision and the reproducibility of the method are good for 15 kinds of bile acid in serum.
Compared with other LC-MS/MS methods, the method has higher sensitivity, simple pretreatment, only one-step protein precipitation treatment, small sample dosage, more detection types, capability of simultaneously detecting 15 bile acids within 6.5 minutes, and capability of being used for clinical diagnosis and health assessment of the bile acids in serum.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A kit for detecting 15 kinds of bile acids in serum is characterized in that:
the 15 bile acids include: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid;
the kit comprises: eluent, calibrator solution, mixed internal standard working solution, protein precipitator and quality control product;
the eluent comprises an eluent A and an eluent B, wherein the eluent A is 0.01-0.1% formic acid-water solution, and the eluent B is methanol;
the calibrator solution comprises a mixed solution of cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid, which are prepared by a blank serum substrate and have known series concentrations;
the mixed internal standard working solution is a mixed solution of cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and taurodeoxycholic acid-d 4, which have known concentration and are prepared by methanol;
the quality control product comprises a mixed solution of cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid, which have known concentration and are prepared by a blank serum substrate.
2. The kit of claim 1, wherein: the eluent A is 0.01 percent formic acid-water solution.
3. The kit of claim 1, wherein: the protein precipitant is methanol.
4. The kit of claim 1, wherein: the blank serum matrix is 5% bovine serum albumin water solution.
5. The kit of claim 1, wherein: the standard solution comprises mixed liquor with the following 7 concentration ratios:
mixed solution 1: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid are present in concentrations of cholic acid 8ng/mL, glycocholic acid 8ng/mL, taurocholic acid 0.8ng/mL, lithocholic acid 0.4ng/mL, glycolithocholic acid 0.4ng/mL, taurocholic acid 0.08ng/mL, deoxycholic acid 8ng/mL, glycodeoxycholic acid 4ng/mL, taurodeoxycholic acid 0.8ng/mL, chenodeoxycholic acid 12ng/mL, glycochenodeoxycholic acid 16ng/mL, taurodeoxycholic acid 4ng/mL, ursodeoxycholic acid 4ng/mL, glycodeoxycholic acid 4ng/, 0.16ng/mL tauro-ursodeoxycholic acid;
and (2) mixed solution: the concentrations of cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid are respectively 20ng/mL of cholic acid, 20ng/mL of glycocholic acid, 2ng/mL of taurocholic acid, 1ng/mL of lithocholic acid, 1ng/mL of glycolithocholic acid, 0.2ng/mL of taurochenodeoxycholic acid, 20ng/mL of deoxycholic acid, 10ng/mL of glycodeoxycholic acid, 2ng/mL of taurodeoxycholic acid, 30ng/mL of chenodeoxycholic acid, 40ng/mL of glycochenodeoxycholic acid, 10ng/mL of taurochenodeoxycholic acid, 10ng/, 0.4ng/mL of tauroursodeoxycholic acid;
and (3) mixed solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid are used at concentrations of, respectively, cholic acid 40ng/mL, glycocholic acid 40ng/mL, taurocholic acid 4ng/mL, lithocholic acid 2ng/mL, glycolithocholic acid 2ng/mL, taurochenodeoxycholic acid 0.4ng/mL, deoxycholic acid 40ng/mL, glycodeoxycholic acid 20ng/mL, taurodeoxycholic acid 4ng/mL, chenodeoxycholic acid 60ng/mL, glycochenodeoxycholic acid 80ng/mL, taurochenodeoxycholic acid 20ng/mL, deoxycholic acid 20ng/mL, glycourso, 0.8ng/mL of tauroursodeoxycholic acid;
and (4) mixed solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid and tauroursodeoxycholic acid are respectively in the concentrations of 100ng/mL of cholic acid, 100ng/mL of glycocholic acid, 10ng/mL of taurocholic acid, 5ng/mL of lithocholic acid, 5ng/mL of glycolithocholic acid, 1ng/mL of taurochenodeoxycholic acid, 100ng/mL of deoxycholic acid, 50ng/mL of glycodeoxycholic acid, 10ng/mL of taurodeoxycholic acid, 150ng/mL of chenodeoxycholic acid, 200ng/mL of glycochenodeoxycholic acid, 50ng/mL of taurochenodeoxycholic acid, 50ng, 2ng/mL of tauroursodeoxycholic acid;
and (5) mixed solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid are respectively at a concentration of cholic acid 200ng/mL, glycocholic acid 200ng/mL, taurocholic acid 20ng/mL, lithocholic acid 10ng/mL, glycolithocholic acid 10ng/mL, taurochenodeoxycholic acid 2ng/mL, deoxycholic acid 200ng/mL, glycodeoxycholic acid 100ng/mL, taurodeoxycholic acid 20ng/mL, chenodeoxycholic acid 300ng/mL, glycochenodeoxycholic acid 400ng/mL, taurochenodeoxycholic acid 100ng/mL, ursodeoxycholic acid 100ng/mL, glycoursodeoxyc, 4ng/mL tauroursodeoxycholic acid;
and (6) mixed solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid are used at concentrations of cholic acid 500ng/mL, glycocholic acid 500ng/mL, taurocholic acid 50ng/mL, lithocholic acid 25ng/mL, glycolithocholic acid 25ng/mL, taurochenodeoxycholic acid 5ng/mL, deoxycholic acid 500ng/mL, glycodeoxycholic acid 250ng/mL, taurodeoxycholic acid 50ng/mL, chenodeoxycholic acid 750ng/mL, glycochenodeoxycholic acid 1000ng/mL, taurochenodeoxycholic acid 250ng/mL, ursodeoxycholic acid 250ng/mL, glycoursodeoxycholic, 10ng/mL tauro-ursodeoxycholic acid;
and (3) mixed solution 7: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, and tauroursodeoxycholic acid are used at concentrations of 1000ng/mL of cholic acid, 1000ng/mL of glycocholic acid, 100ng/mL of taurocholic acid, 50ng/mL of lithocholic acid, 50ng/mL of glycolithocholic acid, 10ng/mL of taurochenodeoxycholic acid, 1000ng/mL of deoxycholic acid, 500ng/mL of glycodeoxycholic acid, 100ng/mL of taurodeoxycholic acid, 1500ng/mL of chenodeoxycholic acid, 2000ng/mL of glycochenodeoxycholic acid, 500ng/mL of taurochenodeoxycholic acid, 500ng, Tauroursodeoxycholic acid 20 ng/mL.
6. The kit of claim 1, wherein: the concentration of cholic acid-d 4, glycocholic acid-d 5, taurocholic acid-d 5, lithocholic acid-d 4, glycolithocholic acid-d 4, taurocholic acid-d 5, deoxycholic acid-d 4, glycodeoxycholic acid-d 5, taurodeoxycholic acid-d 4, chenodeoxycholic acid-d 4, glycochenodeoxycholic acid-d 4, taurochenodeoxycholic acid-d 5, ursodeoxycholic acid-d 4, glycoursodeoxycholic acid-d 4 and tauroursodeoxycholic acid-4 in the mixed internal standard working solution is 50ng/mL cholic acid-d 4, 50ng/mL glycocholic acid-d 5, 5/mL taurocholic acid-d 5, 2.5ng/mL cholic acid-d 4, 2.5ng/mL glycolithocholic acid-d 4, 0.5ng/mL taurocholic acid-d 5, 50ng/mL glycocholic acid-d 4mL, 25ng/mL glycodeoxycholic acid-d 5, 5ng/mL taurodeoxycholic acid-d 4, 75ng/mL chenodeoxycholic acid-d 4, 100ng/mL glycochenodeoxycholic acid-d 4, 25ng/mL taurochenodeoxycholic acid-d 5, 25ng/mL ursodeoxycholic acid-d 4, 25ng/mL glycoursodeoxycholic acid-d 4, and 1ng/mL taurodeoxycholic acid-d 4.
7. The kit of claim 1, wherein: the quality control product comprises mixed liquid with low, medium and high concentrations:
low concentration quality control solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid are respectively in the concentrations of 20ng/mL, 20ng/mL glycocholic acid, 2ng/mL taurocholic acid, 1ng/mL lithocholic acid, 1ng/mL glycolithocholic acid, 0.2ng/mL taurochenodeoxycholic acid, 20ng/mL deoxycholic acid, 10ng/mL glycodeoxycholic acid, 2ng/mL taurodeoxycholic acid, 30ng/mL chenodeoxycholic acid, 40ng/mL glycochenodeoxycholic acid, 10ng/mL taurochenodeoxycholic acid, 10ng/mL deoxycholic acid, 10ng/mL glycoursodeoxycholic, 0.4ng/mL of tauroursodeoxycholic acid;
medium concentration quality control solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid are respectively 100ng/mL, glycocholic acid 100ng/mL, taurocholic acid 10ng/mL, lithocholic acid 5ng/mL, glycolithocholic acid 5ng/mL, taurocholic acid 1ng/mL, deoxycholic acid 100ng/mL, glycodeoxycholic acid 50ng/mL, taurodeoxycholic acid 10ng/mL, chenodeoxycholic acid 150ng/mL, glycochenodeoxycholic acid 200ng/mL, taurochenodeoxycholic acid 50ng/mL, ursodeoxycholic acid 50ng/mL, glycoursodeoxycholic acid 50ng/mL, taurode, 2ng/mL of tauroursodeoxycholic acid;
high concentration quality control solution: cholic acid, glycocholic acid, taurocholic acid, lithocholic acid, glycolithocholic acid, taurodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid are respectively 400ng/mL, glycocholic acid 400ng/mL, taurocholic acid 40ng/mL, lithocholic acid 20ng/mL, glycolithocholic acid 20ng/mL, taurochenodeoxycholic acid 4ng/mL, deoxycholic acid 400ng/mL, glycodeoxycholic acid 200ng/mL, taurodeoxycholic acid 40ng/mL, chenodeoxycholic acid 600ng/mL, glycochenodeoxycholic acid 800ng/mL, taurochenodeoxycholic acid 200ng/mL, ursodeoxycholic acid 200ng/mL, glycoursodeoxycholic acid 200ng/mL, 8ng/mL tauroursodeoxycholic acid.
8. Use of the kit of any one of claims 1 to 6 for detecting bile acids in serum using ultra performance liquid chromatography tandem mass spectrometry.
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| CN114383911A (en) * | 2022-01-21 | 2022-04-22 | 江苏省中医院 | Application of glycodeoxycholic acid in preparation of kit for diagnosing heart failure |
| CN114432462A (en) * | 2022-02-25 | 2022-05-06 | 复旦大学 | Metabolite for predicting and treating obesity and/or diabetes and preparation thereof |
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| CN114778737A (en) * | 2022-04-27 | 2022-07-22 | 天津国科医工科技发展有限公司 | Liquid chromatography detection sample pretreatment method capable of shortening time |
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