Kit for detecting 15 bile acid spectra and three bile acid precursors in serum simultaneously and detection method therefor

Abstract

Disclosed in the present invention are a kit for detecting 15 bile acid profiles and three bile acid precursors in serum simultaneously and a detection method therefor. The 15 bile acid profiles comprise: 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 taurolithocholic acid; and the three bile acid precursors comprise: 3α,7α-dihydroxycoprostanic acid, 3α,7α,12α-trihydroxycholestanoic acid and 7α-hydroxy-4-cholesten-3-one. The kit and detection method of the present invention enable the simultaneous and accurate detection of the 15 bile acid profiles and the three bile acid precursors, with a short detection time and a high detection accuracy. This, to a certain extent, meets the clinical need for assessing the enterohepatic cycle composed of the biliary system, intestines, portal venous circulation and hepatocytes.

Description

A kit and detection method for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum. Technical Field

[0001] This invention relates to the field of metabolomics technology, specifically to a kit and detection method for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum. Background Technology

[0002] Bile acids are a collective term for a class of cholanonic acids and are the main organic components of bile. They are derived from cholesterol in hepatocytes and are the final products of cholesterol catabolism. As active components of bile, they are secreted from the liver into the intestinal lumen, playing a crucial role in the emulsification, subsequent absorption, and transport of dietary fats, drugs, and fat-soluble vitamins in the intestine. Furthermore, bile acids can act as signaling molecules and metabolic regulators, modulating host lipid, glucose, and cholesterol homeostasis by activating the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor (TGR5).

[0003] As endogenous metabolites in the human body, bile acids are diverse, exhibiting varied and even contradictory biochemical and physiological properties due to differences in their side chain structure, stereochemical properties, and the number and position of hydroxyl groups. For example, cholic acid (CA) promotes intestinal cholesterol adsorption, while chenodeoxycholic acid (CDCA) inhibits it; lithocholic acid (LCA) and deoxycholic acid (DCA) are considered potential carcinogens, while ursodeoxycholic acid (UDCA) has certain anti-cancer effects.

[0004] 7α-Hydroxy-4-cholesten-3-one (C4) is an oxidase product of cholesterol metabolism via cholesterol 7α-hydroxylase (also known as cholesterol 7-α-monooxygenase or the enzyme of cytochrome P450 7A1 (CYP7A1)). C4 is a stable intermediate in the rate-limiting pathway of bile acid biosynthesis. Plasma C4 levels are correlated with CYP7A1 enzyme activity and can serve as a biomarker for bile acid synthesis.

[0005] The conversion of cholesterol into primary bile acids chenodeoxycholic acid and bile acids involves hydroxylation of the steroid nucleus and oxidative shortening of the side chain. Intermediates in this process are the C27 bile acid intermediates 3α,7α-dihydroxycobalamin prostaglandin (DHCA) and 3α,7α,12α-trihydroxycholestannic acid (THCA). The primary metabolic pathways of DHCA and THCA involve chain shortening via a series of peroxisome β-oxidation reactions catalyzed by a group of enzymes that specifically act on 2-methyl-substituted fatty acids. Patients with peroxisome β-oxidation defects, due to peroxisome biogenesis or impaired specificity of bile acid β-oxidases, accumulate DHCA and THCA in their blood and bile. Therefore, the measurement of DHCA and THCA in body fluids is an important marker for diagnosing postpartum and prenatal peroxisome disorders.

[0006] Different types of bile acids have significantly different effects on diseases. Currently, clinical diagnosis relies solely on measuring total bile acid levels in human serum, which is prone to missed or misdiagnosed cases due to its poor sensitivity and specificity. Therefore, comprehensive and accurate monitoring of the content of various types of bile acids in different biological samples can help explore new strategies for disease diagnosis, treatment, and prognosis.

[0007] Currently, the main methods for detecting bile acid profiles and bile acid precursor concentrations in the human body include high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Blood samples have a complex matrix, and using HPLC to detect bile acid profiles and bile acid precursors in serum samples requires thorough separation and purification to remove interfering substances. Therefore, the pretreatment process is complex, time-consuming, involves many types of reagents, has high consumable costs, and is time-consuming, making it unsuitable for large-scale clinical sample testing. HPLC-MS / MS combines the advantages of chromatographic separation with the high sensitivity, specificity, and strong anti-interference capabilities of mass spectrometry, making it the "gold standard" for detecting small molecule compounds.

[0008] Existing methods for detecting bile acid profiles can only detect bile acid cycle markers, lacking methods that can simultaneously and effectively detect bile acid synthesis markers such as 7α-hydroxy-4-cholesten-3-one (C4), 3α,7α-dihydroxycobalamin prostaglandin (DHCA), and 3α,7α,12α-trihydroxycholestannic acid (THCA). This results in a gap in the assessment of the enterohepatic cycle, which consists of the biliary system, intestine, portal circulation, and hepatocytes.

[0009] This invention simultaneously detects 15 bile acids and 3 bile acid precursors in serum, using 50 μL of venous serum as the test sample, and employs liquid chromatography-mass spectrometry (LC-MS / MS). Pretreatment involves a simple protein precipitation method for separation and extraction from dried serum. The pretreatment procedure is simple, the sample detection time is short, the detection accuracy is high, and the sample volume is small, thus addressing, to some extent, the clinical need for assessing the enterohepatic cycle, which comprises the biliary system, intestine, portal circulation, and hepatocytes. Summary of the Invention

[0010] The purpose of this invention is to provide a kit and method for simultaneously detecting the concentrations of 15 bile acids and 3 bile acid precursors in serum based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). After collecting venous serum, interference is removed by protein precipitation followed by dilution, and the analytes are extracted. Quantitative detection is then performed using LC-MS / MS to determine the concentrations of the 15 bile acids and 3 bile acid precursors in the human body. The standard curve used in the detection process is a blank serum prepared through repeated freeze-thaw cycles and suspension adsorption, which effectively reduces matrix effects and improves detection accuracy.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] <First Aspect>

[0013] This invention provides a kit for simultaneously detecting the concentrations of 15 bile acids and 3 bile acid precursors in serum. The 15 bile acids include: cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, and taurocholic acid.

[0014] The three bile acid precursors include: 3α,7α-dihydroxycobalt prostatic acid, 3α,7α,12α-trihydroxycholestan acid, and 7α-hydroxy-4-cholesterol-3-one;

[0015] The kit includes: standard curve solution, quality control materials, protein internal standard precipitant, and mobile phase;

[0016] The protein internal standard precipitant includes an internal standard stock solution, which is a mixed solution containing isotopic internal standards of the 15 bile acid profiles and 3 bile acid precursors;

[0017] The standard curve solution is prepared by mixing a blank serum matrix with the standard curve working solution. The standard curve working solution includes a mixed solution of the 15 bile acid spectra and 3 bile acid precursors (with known concentrations).

[0018] The quality control material is prepared by mixing a blank serum matrix with a quality control working solution. The quality control working solution includes a mixed solution of the 15 bile acid profiles and 3 bile acid precursors (with known concentrations).

[0019] The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.01-0.1% aqueous solution of formic acid and mobile phase B is an acetonitrile solution.

[0020] In one embodiment of the present invention, the mobile phase A is a 0.01% aqueous solution of formic acid.

[0021] As one embodiment of the present invention, the protein internal standard precipitant is prepared by internal standard stock solution and acetonitrile, wherein the volume ratio of internal standard stock solution to acetonitrile is 1:15 to 1:20.

[0022] As one embodiment of the present invention, the internal standard stock solution is prepared by mixing acetonitrile with the isotopic internal standards of the 15 bile acid spectra and 3 bile acid precursors.

[0023] As one embodiment of the present invention, the isotopic internal standards of the 15 bile acid spectra include cholic acid-d4, deoxycholic acid-d4, chenodeoxycholic acid-d4, ursodeoxycholic acid-d4, lithocholic acid-d4, glycocholic acid-d4, glycodeoxycholic acid-d4, glycochenodeoxycholic acid-d4, glycoursodeoxycholic acid-d4, glycolithocholic acid-d4, taurocholic acid-d4, taurodeoxycholic acid-d4, taurochenodeoxycholic acid-d4, and tauroursodeoxycholic acid-d4.

[0024] The isotopic internal standards for the three bile acid precursors include 3α,7α-dihydroxycobalt prostaglandin-d3, 3α,7α,12α-trihydroxycholestannic acid-d3, and 7α-hydroxy-4-cholesterol-3-one-d7.

[0025] As one embodiment of the present invention, the blank serum matrix is ​​prepared by: using heparin-anticoagulated normal blank serum, repeatedly freezing and thawing 5 to 10 times, adding activated carbon to prepare a serum suspension, vortexing to allow it to fully adsorb, centrifuging, taking the supernatant and filtering to obtain the blank serum matrix.

[0026] Furthermore, the concentration of activated charcoal in the serum suspension is 0.05–0.2 g / mL;

[0027] And / or, the temperature of the vortex is 20–30°C, and the time is 1–3 hours;

[0028] And / or, the centrifugation speed is 12000-13000 RPM, the temperature is 3-6℃, and the time is 5-20 min.

[0029] Preferably, the concentration of activated charcoal in the serum suspension is 0.2 g / mL;

[0030] And / or, the temperature of the vortex is room temperature (25°C) and the time is 2 hours;

[0031] And / or, the centrifugation speed is 13000 RPM, the temperature is 4°C, and the time is 20 min.

[0032] Preferably, the number of freeze-thaw cycles is 5.

[0033] In one embodiment of the present invention, the standard curve solution comprises a mixture of the following six concentration ratios:

[0034] S1, 10 ng / ml cholic acid, 10 ng / ml deoxycholic acid, 10 ng / ml chenodeoxycholic acid, 10 ng / ml ursodeoxycholic acid, 2.5 ng / ml lithocholic acid, 20 ng / ml glycocholic acid, 10 ng / ml glycodeoxycholic acid, 20 ng / ml glycochenodeoxycholic acid, 10 ng / ml ursodeoxycholic acid, 10 ng / ml glycolithocholic acid, 10 ng / ml taurocholic acid, 2.5 ng / ml taurodeoxycholic acid, 2.5 ng / ml taurochenodeoxycholic acid, 2.5 ng / ml taurochenodeoxycholic acid, 2.5 ng / ml taurochenodeoxycholic acid, 2.5 ng / ml taurochenodeoxycholic acid, 2.5 ng / ml 3α,7α-DHCA, 20 ng / ml 3α,7α,12α-THCA and 10 ng / ml C4;

[0035] S2, 20 ng / ml cholic acid, 20 ng / ml deoxycholic acid, 20 ng / ml chenodeoxycholic acid, 20 ng / ml ursodeoxycholic acid, 5 ng / ml lithocholic acid, 40 ng / ml glycocholic acid, 20 ng / ml glycodeoxycholic acid, 40 ng / ml glycochenodeoxycholic acid, 20 ng / ml ursodeoxycholic acid, 20 ng / ml glycolithocholic acid, 20 ng / ml taurocholic acid, 5 ng / ml taurodeoxycholic acid, 5 ng / ml taurochenodeoxycholic acid, 5 ng / ml taurochenodeoxycholic acid, 5 ng / ml taurochenodeoxycholic acid, 5 ng / ml taurochenodeoxycholic acid, 5 ng / ml taurochenodeoxycholic acid, 5 ng / ml 3α,7α-DHCA, 40 ng / ml 3α,7α,12α-THCA and 20 ng / ml C4;

[0036] S3, 50 ng / ml cholic acid, 50 ng / ml deoxycholic acid, 50 ng / ml chenodeoxycholic acid, 50 ng / ml ursodeoxycholic acid, 12.5 ng / ml lithocholic acid, 100 ng / ml glycocholic acid, 50 ng / ml glycodeoxycholic acid, 100 ng / ml glycochenodeoxycholic acid, 50 ng / ml ursodeoxycholic acid, 50 ng / ml glycolithocholic acid, 50 ng / ml taurocholic acid, 12.5 ng / ml taurodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurolithocholic acid, 12.5 ng / ml 3α,7α-DHCA, 100 ng / ml 3α,7α,12α-THCA and 50 ng / ml C4;

[0037] S4, 100 ng / ml cholic acid, 100 ng / ml deoxycholic acid, 100 ng / ml chenodeoxycholic acid, 100 ng / ml ursodeoxycholic acid, 25 ng / ml lithocholic acid, 200 ng / ml glycocholic acid, 100 ng / ml glycodeoxycholic acid, 200 ng / ml glycochenodeoxycholic acid, 100 ng / ml ursodeoxycholic acid, 100 ng / ml glycolithocholic acid, 100 ng / ml taurocholic acid, 25 ng / ml taurodeoxycholic acid, 25 ng / ml taurochenodeoxycholic acid, 25 ng / ml taurochenodeoxycholic acid, 25 ng / ml taurochenodeoxycholic acid, 25 ng / ml taurochenodeoxycholic acid, 25 ng / ml taurochenodeoxycholic acid, 25 ng / ml 3α,7α-DHCA, 200 ng / ml 3α,7α,12α-THCA and 100 ng / ml C4;

[0038] S5, 500 ng / ml cholic acid, 500 ng / ml deoxycholic acid, 500 ng / ml chenodeoxycholic acid, 500 ng / ml ursodeoxycholic acid, 125 ng / ml lithocholic acid, 1000 ng / ml glycocholic acid, 500 ng / ml glycodeoxycholic acid, 1000 ng / ml glycochenodeoxycholic acid, 500 ng / ml ursodeoxycholic acid, 500 ng / ml glycolithocholic acid, 500 ng / ml taurocholic acid, 125 ng / ml taurodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurolithocholic acid, 125 ng / ml 3α,7α-DHCA, 1000 ng / ml 3α,7α,12α-THCA and 500 ng / ml C4;

[0039] S6, 2000 ng / ml cholic acid, 2000 ng / ml deoxycholic acid, 2000 ng / ml chenodeoxycholic acid, 2000 ng / ml ursodeoxycholic acid, 500 ng / ml lithocholic acid, 4000 ng / ml glycocholic acid, 2000 ng / ml glycodeoxycholic acid, 4000 ng / ml glycochenodeoxycholic acid, 2000 ng / ml ursodeoxycholic acid, 2000 ng / ml glycolithocholic acid, 2000 ng / ml taurocholic acid, 500 ng / ml taurodeoxycholic acid, 500 ng / ml taurochenodeoxycholic acid, 500 ng / ml tauroursodeoxycholic acid, 500 ng / ml taurolithocholic acid, 500 ng / ml 3α,7α-DHCA, 4000 ng / ml 3α,7α,12α-THCA and 2000 ng / ml C4.

[0040] As one embodiment of the present invention, the standard curve solution is prepared by mixing blank serum matrix and standard curve working solution diluted 10 times at a volume ratio of 99:1, and the blank serum matrix is ​​prepared from heparin-anticoagulated normal pig blank serum.

[0041] As one embodiment of the present invention, the standard curve working solution comprises a mixture of the following six concentration ratios:

[0042] W1 (Mixed Solution 1): 10 μg / ml cholic acid, 10 μg / ml deoxycholic acid, 10 μg / ml chenodeoxycholic acid, 10 μg / ml ursodeoxycholic acid, 2.5 μg / ml lithocholic acid, 20 μg / ml glycocholic acid, 10 μg / ml glycodeoxycholic acid, 20 μg / ml glycochenodeoxycholic acid, 10 μg / ml ursodeoxycholic acid, 10 μg / ml glycolithocholic acid, 10 μg / ml taurocholic acid, 2.5 μg / ml taurocholic acid, 2.5 μg / ml taurocholic acid, 2.5 μg / ml taurocholic acid, 2.5 μg / ml taurocholic acid, 2.5 μg / ml 3α,7α-DHCA, 20 μg / ml 3α,7α,12α-THCA and 10 μg / ml C4;

[0043] W2 (Mixed Solution 2): 20 μg / ml cholic acid, 20 μg / ml deoxycholic acid, 20 μg / ml chenodeoxycholic acid, 20 μg / ml ursodeoxycholic acid, 5 μg / ml lithocholic acid, 40 μg / ml glycinecholic acid, 20 μg / ml glycine deoxycholic acid, 40 μg / ml glycine chenodeoxycholic acid, 20 μg / ml ursodeoxycholic acid, 20 μg / ml glycine lithocholic acid, 20 μg / ml taurocholic acid, 5 μg / ml taurocholic acid, 5 μg / ml taurocholic acid, 5 μg / ml taurocholic acid, 5 μg / ml taurocholic acid, 5 μg / ml 3α,7α-DHCA, 40 μg / ml 3α,7α,12α-THCA and 20 μg / ml C4;

[0044] W3 (Mixed Solution 3): 50 μg / ml cholic acid, 50 μg / ml deoxycholic acid, 50 μg / ml chenodeoxycholic acid, 50 μg / ml ursodeoxycholic acid, 12.5 μg / ml lithocholic acid, 100 μg / ml glycocholic acid, 50 μg / ml glycodeoxycholic acid, 100 μg / ml glycochenodeoxycholic acid, 50 μg / ml ursodeoxycholic acid, 50 μg / ml glycocholic acid Lithocholic acid, 50 μg / ml taurine, 12.5 μg / ml taurine deoxycholic acid, 12.5 μg / ml taurine chenodeoxycholic acid, 12.5 μg / ml taurine ursodeoxycholic acid, 12.5 μg / ml taurine lithocholic acid, 12.5 μg / ml 3α,7α-DHCA, 100 μg / ml 3α,7α,12α-THCA and 50 μg / ml C4;

[0045] W4 (Mixed Solution 4): 100 μg / ml cholic acid, 100 μg / ml deoxycholic acid, 100 μg / ml chenodeoxycholic acid, 100 μg / ml ursodeoxycholic acid, 25 μg / ml lithocholic acid, 200 μg / ml glycocholic acid, 100 μg / ml glycodeoxycholic acid, 200 μg / ml glycochenodeoxycholic acid, 100 μg / ml ursodeoxycholic acid, 100 μg / ml glycolithocholic acid, 100μg / ml taurine cholic acid, 25μg / ml taurine deoxycholic acid, 25μg / ml taurine chenodeoxycholic acid, 25μg / ml taurine ursodeoxycholic acid, 25μg / ml taurine lithocholic acid, 25μg / ml 3α,7α-DHCA, 200μg / ml 3α,7α,12α-THCA and 100μg / ml C4;

[0046] W5 (Mixed Solution 5): 500 μg / ml cholic acid, 500 μg / ml deoxycholic acid, 500 μg / ml chenodeoxycholic acid, 500 μg / ml ursodeoxycholic acid, 125 μg / ml lithocholic acid, 1000 μg / ml glycocholic acid, 500 μg / ml glycodeoxycholic acid, 1000 μg / ml glycochenodeoxycholic acid, 500 μg / ml ursodeoxycholic acid, 500 μg / ml glycine lithocholic acid, 500μg / ml taurine cholic acid, 125μg / ml taurine deoxycholic acid, 125μg / ml taurine chenodeoxycholic acid, 125μg / ml taurine ursodeoxycholic acid, 125μg / ml taurine lithocholic acid, 125μg / ml 3α,7α-DHCA, 1000μg / ml 3α,7α,12α-THCA and 500μg / ml C4;

[0047] W6 (Mixed Solution 6): 2000 μg / ml cholic acid, 2000 μg / ml deoxycholic acid, 2000 μg / ml chenodeoxycholic acid, 2000 μg / ml ursodeoxycholic acid, 500 μg / ml lithocholic acid, 4000 μg / ml glycocholic acid, 2000 μg / ml glycodeoxycholic acid, 4000 μg / ml glycochenodeoxycholic acid, 2000 μg / ml ursodeoxycholic acid, 200 0 μg / ml glycolithocholic acid, 2000 μg / ml taurine cholic acid, 500 μg / ml taurine deoxycholic acid, 500 μg / ml taurine chenodeoxycholic acid, 500 μg / ml taurine ursodeoxycholic acid, 500 μg / ml taurine lithocholic acid, 500 μg / ml 3α,7α-DHCA, 4000 μg / ml 3α,7α,12α-THCA and 2000 μg / ml C4.

[0048] In one specific embodiment of the present invention, the quality control materials include quality control material QL and quality control material QH, and the concentration ratio of the two quality control materials is as follows:

[0049] Quality control products (QL): 50 ng / ml cholic acid, 50 ng / ml deoxycholic acid, 50 ng / ml chenodeoxycholic acid, 50 ng / ml ursodeoxycholic acid, 12.5 ng / ml lithocholic acid, 100 ng / ml glycocholic acid, 50 ng / ml glycodeoxycholic acid, 100 ng / ml glycochenodeoxycholic acid, 50 ng / ml ursodeoxycholic acid, 50 ng / ml glycolithocholic acid, 50 ng / ml taurocholic acid, 12.5 ng / ml taurodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurochenodeoxycholic acid, 12.5 ng / ml taurolithocholic acid, 12.5 ng / ml 3α,7α-DHCA, 100 ng / ml 3α,7α,12α-THCA and 50 ng / ml C4;

[0050] Quality control products (QH): 500 ng / ml cholic acid, 500 ng / ml deoxycholic acid, 500 ng / ml chenodeoxycholic acid, 500 ng / ml ursodeoxycholic acid, 125 ng / ml lithocholic acid, 1000 ng / ml glycocholic acid, 500 ng / ml glycodeoxycholic acid, 1000 ng / ml glycochenodeoxycholic acid, 500 ng / ml ursodeoxycholic acid, 500 ng / ml glycolithocholic acid, 500 ng / ml taurocholic acid, 125 ng / ml taurodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml taurochenodeoxycholic acid, 125 ng / ml 3α,7α-DHCA, 1000 ng / ml 3α,7α,12α-THCA, and 500 ng / ml C4.

[0051] In one specific embodiment of the present invention, the quality control product is prepared by mixing blank serum matrix and quality control product working solution at a volume ratio of 99:1, and the blank serum matrix is ​​prepared from heparin-anticoagulated normal human blank serum.

[0052] As a specific embodiment of the present invention, the quality control working solution includes two concentration ratios of QL and QH, and the concentration ratios of the two quality control working solutions are as follows:

[0053] Quality control working solutions (QL): 5 μg / ml cholic acid, 5 μg / ml deoxycholic acid, 5 μg / ml chenodeoxycholic acid, 5 μg / ml ursodeoxycholic acid, 1.25 μg / ml lithocholic acid, 10 μg / ml glycocholic acid, 5 μg / ml glycodeoxycholic acid, 10 μg / ml glycochenodeoxycholic acid, 5 μg / ml ursodeoxycholic acid, 5 μg / ml glycolithocholic acid, 5 μg / ml taurocholic acid, 1.25 μg / ml taurocholic acid, 1.25 μg / ml taurocholic acid, 1.25 μg / ml taurocholic acid, 1.25 μg / ml taurocholic acid, 1.25 μg / ml 3α,7α-DHCA, 10 μg / ml 3α,7α,12α-THCA, and 5 μg / ml C4;

[0054] Quality control working solution QH: 50 μg / ml cholic acid, 50 μg / ml deoxycholic acid, 50 μg / ml chenodeoxycholic acid, 50 μg / ml ursodeoxycholic acid, 12.5 μg / ml lithocholic acid, 100 μg / ml glycocholic acid, 50 μg / ml glycodeoxycholic acid, 100 μg / ml glycochenodeoxycholic acid, 50 μg / ml ursodeoxycholic acid, 50 μg / ml glycocalycate. Lithocholic acid, 50 μg / ml taurine, 12.5 μg / ml taurine deoxycholic acid, 12.5 μg / ml taurine chenodeoxycholic acid, 12.5 μg / ml taurine ursodeoxycholic acid, 12.5 μg / ml taurine lithocholic acid, 12.5 μg / ml 3α,7α-DHCA, 100 μg / ml 3α,7α,12α-THCA and 50 μg / ml C4.

[0055] <Second aspect>

[0056] This invention provides a method for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum, using the kit described in any of the above-mentioned claims.

[0057] As one embodiment of the present invention, the method includes the following steps:

[0058] (1) Take a serum sample, add protein internal standard precipitant, and shake to mix;

[0059] (2) After centrifugation, the supernatant was taken, diluted, and then detected by liquid chromatography-tandem mass spectrometry.

[0060] (3) Calculate the concentrations of 15 bile acids and 3 bile acid precursors in serum samples based on the standard curve.

[0061] As one embodiment of the present invention, the chromatographic column used in step (2) for on-machine detection is a Waters UPLC BEH C18 chromatographic column; the column temperature of the chromatographic column is 40-45℃ and the flow rate is 0.28-0.33mL / min.

[0062] Preferably, the column temperature of the chromatographic column is 45°C and the flow rate is 0.3 mL / min.

[0063] Furthermore, the chromatographic column has the specifications of UPLC BEH C182.1*100mm and a pore size of 1.7μm.

[0064] Furthermore, the sample injection volume during the on-machine testing is 5 μL.

[0065] As one embodiment of the present invention, in step (2), during the on-machine detection, the gradient elution procedure is as follows:

[0066] As one embodiment of the present invention, the method for plotting the standard curve in step (3) is as follows: take the standard curve solution, add the protein internal standard precipitant, mix well, centrifuge and take the supernatant, dilute and then test it on the instrument. Plot the standard curve with the concentration of the standard curve solution as the abscissa and the ratio of the peak area to the internal standard peak area as the ordinate.

[0067] As some specific embodiments of the present invention, the standard curve solution is prepared by the following method:

[0068] a. Preparation of blank serum matrix: Heparin-anticoagulated normal porcine serum was repeatedly frozen and thawed 5 times, and activated carbon was added to prepare a serum suspension with an activated carbon concentration of 0.05-0.2 g / mL. The suspension was vortexed at room temperature for 1-3 hours to ensure sufficient contact and adsorption, and then centrifuged at 12000-13000 rpm at 4℃ for 5-20 minutes. The supernatant was then filtered to obtain blank serum.

[0069] b. Preparation of standard curve solution: The diluted standard curve working solution and the blank serum matrix prepared in the above steps are mixed at a volume ratio of 1:99 to prepare the standard curve solution.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] 1) Comprehensive and effective detection of bile acid synthesis and metabolism: It can accurately detect 15 bile acid profiles (CA, DCA, CDCA, UDCA, LCA, TCA, TLCA, TDCA, TCDCA, TUDCA, GCA, GDCA, GLCA, GCDCA, GUDCA) and 3 bile acid precursor markers (DHCA, THCA, C4) at the same time, which can effectively help monitor the entire metabolic pathway of bile acid synthesis and absorption. At present, there is no method on the market that can simultaneously detect these 3 bile acid precursors and 15 bile acid profiles. This invention can provide support for the auxiliary diagnosis of bile acid synthesis disorders and bile acid metabolism abnormalities.

[0072] 2) Simple pretreatment process: Sample pretreatment uses protein precipitation and ultrasonic-assisted extraction of analytes. After centrifugation, the supernatant can be used for instrumental detection. Compared with liquid-liquid extraction and solid-liquid extraction, the operation steps are fewer and more convenient and simple.

[0073] 3) Short effective detection cycle: Using an effective mobile phase with a reasonable gradient, and switching between positive and negative ion modes, the detection time is effectively shortened to 16 minutes; rapid separation and detection of 15 bile acid spectra and 3 bile acid precursors;

[0074] 4) This invention utilizes repeated freeze-thaw temperature changes to destroy endogenous active substances and sedimented impurities in heparin-anticoagulated normal serum, and combines this with suspension adsorption technology for effective adsorption, thereby effectively preparing a matrix blank serum background that approximates the type of test sample and effectively reducing matrix effects. Attached Figure Description

[0075] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0076] Figure 1 shows the spectra of 15 bile acids and the total ion chromatogram of 3 bile acid precursors extracted from serum samples obtained in Example 2.

[0077] Figures 2 and 3 are the lower limit of detection and the upper limit of detection of bile acid (CA) in Example 2, respectively.

[0078] Figures 4 and 5 show the lower limit of detection and the upper limit of detection of deoxycholic acid (DCA) in Example 2, respectively.

[0079] Figure 6 shows the lower limit of detection for chenodeoxycholic acid (CDCA) in Example 2;

[0080] Figure 7 shows the upper limit of detection for chenodeoxycholic acid (CDCA) in Example 2;

[0081] Figure 8 shows the lower limit of detection for chenodeoxycholic acid (UDCA) in Example 2.

[0082] Figure 9 shows the upper limit of detection for chenodeoxycholic acid (UDCA) in Example 2.

[0083] Figure 10 shows the lower limit of detection for lithocholic acid (LCA) in Example 2;

[0084] Figure 11 shows the upper limit of detection for lithocholic acid (LCA) in Example 2;

[0085] Figure 12 shows the limit of detection for glycocholic acid (GCA) in Example 2.

[0086] Figure 13 shows the upper limit of detection for glycocholic acid (GCA) in Example 2;

[0087] Figure 14 shows the limit of detection for glycine deoxycholic acid (GDCA) in Example 2.

[0088] Figure 15 shows the upper limit of detection for glycine deoxycholic acid (GDCA) in Example 2.

[0089] Figure 16 shows the limit of detection for glycochenodeoxycholic acid (GCDCA) in Example 2.

[0090] Figure 17 shows the upper limit of detection for glycochenodeoxycholic acid (GCDCA) in Example 2.

[0091] Figure 18 shows the limit of detection for glycoursodeoxycholic acid (GUDCA) in Example 2.

[0092] Figure 19 shows the upper limit of detection for glycoursodeoxycholic acid (GUDCA) in Example 2.

[0093] Figure 20 shows the limit of detection for glycolithocholic acid (GLCA) in Example 2.

[0094] Figure 21 shows the upper limit of detection of glycolithocholic acid (GLCA) in Example 2;

[0095] Figure 22 shows the lower limit of detection for taurine (TCA) in Example 2.

[0096] Figure 23 shows the upper limit of detection for taurine (TCA) in Example 2;

[0097] Figure 24 shows the lowest detection limit of TDA in Example 2;

[0098] Figure 25 shows the maximum detection limit of TDA in Example 2.

[0099] Figure 26 shows the lower limit of detection for taurine chenodeoxycholic acid (TCDCA) in Example 2.

[0100] Figure 27 shows the upper limit of detection for taurine chenodeoxycholic acid (TCDCA) in Example 2.

[0101] Figure 28 shows the lower limit of detection for ursodeoxycholic acid (TUDCA) in Example 2.

[0102] Figure 29 shows the upper limit of detection for ursodeoxycholic acid (TUDCA) in Example 2.

[0103] Figure 30 shows the lower limit of detection for taurocholic acid (TLCA) in Example 2.

[0104] Figure 31 shows the upper limit of detection for taurocholic acid (TLCA) in Example 2.

[0105] Figure 32 shows the lower limit of detection for 3α,7α-dihydroxycobalt prostatic acid (DHCA) in Example 2.

[0106] Figure 33 shows the upper limit of detection for 3α,7α-dihydroxycobalt prostaglandin (DHCA) in Example 2.

[0107] Figure 34 shows the limit of detection for 3α,7α,12α-trihydroxycholestannic acid (THCA) in Example 2.

[0108] Figure 35 shows the upper limit of detection for 3α,7α,12α-trihydroxycholestanic acid (THCA) in Example 2.

[0109] Figure 36 shows the limit of detection for 7α-hydroxy-4-cholesterol-3-one-d7(C4) in Example 2;

[0110] Figure 37 shows the upper limit of detection for 7α-hydroxy-4-cholesterol-3-one-d7(C4) in Example 2.

[0111] Figures 38-43 are chromatograms of mobile phase compositions 1-6 in Experimental Example 1. Detailed Implementation

[0112] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0113] Example 1 - Solution Preparation

[0114] 1. Standard curve solution

[0115] 1.1 Prepare standard stock solutions of 15 bile acid profiles and 3 bile acid precursors.

[0116] Eighteen bile acid profiles and precursor standard powders were purchased from the market according to their CAS numbers. After weighing them using a precision balance, each standard was prepared into a standard stock solution using methanol / dimethyl sulfoxide. The concentration and specifications of each standard and the concentration of the prepared standard stock solution are shown in Table 1.

[0117] Table 118 Bile Acid Standard Reference Materials: CAS Numbers and Standard Stock Solutions

[0118] 1.2 Preparation of intermediate mixed liquid

[0119] According to the mixed intermediate solution preparation table shown in Table 2, take the 18 standard stock solutions prepared in 1.1 according to the "volume of standard stock solution added", and add 500 μL of 30% acetonitrile aqueous solution for mixing and dilution to obtain the mixed intermediate solution, which is used to prepare the subsequent "standard curve working solution". The concentration of each standard in the mixed intermediate solution is shown in Table 2.

[0120] Table 2. Preparation of Intermediate Mixture

[0121] 1.3 Preparation of standard curve working solution

[0122] Using the mixed intermediate solution (solution A) prepared in section 1.2 as the stock solution, and 30% acetonitrile aqueous solution as the diluent, the mixed intermediate solution was diluted according to the volumes shown in Table 3 to prepare three standard curve working solutions: W6, W5, and W4. Then, using standard curve working solution W4 as the stock solution, and 30% acetonitrile aqueous solution as the diluent, the W4 standard curve working solution was diluted according to the volumes shown in Table 3 to prepare three standard curve working solutions: W3, W2, and W1. The theoretical concentrations of each standard in standard curve working solutions W1 through W6 are shown in Table 4.

[0123] Table 3. Preparation of Standard Curve Working Solution

[0124] Table 4. Concentration of working solution for standard curve

[0125] 1.4 Preparation of standard curve solution

[0126] a. Blank serum matrix: Heparin-anticoagulated normal porcine blank serum was repeatedly frozen and thawed 5 times, and then activated carbon was added to prepare a serum suspension with an activated carbon concentration of 0.2 g / mL. The suspension was vortexed at room temperature for 2 h to ensure sufficient contact and adsorption, and then centrifuged at 13000 rpm and 4℃ for 20 min. The supernatant was then filtered to obtain the blank serum required for this experiment.

[0127] b. Preparation of standard curve solutions: Take the standard curve working solutions (W1 to W6) prepared in 1.3, dilute them 10 times with 15% acetonitrile aqueous solution, and then prepare standard curve solutions with blank serum at a ratio of 1:99 (V:V) to obtain standard curve solutions S1 to S6. The theoretical concentrations of each standard in the standard curve solutions are shown in Table 5.

[0128] Taking the S1 standard curve solution as an example: Take 10 μL of W1 standard curve working solution and slowly add it to 90 μL of 15% acetonitrile aqueous solution. After mixing, take 10 μL of W1 standard curve working solution diluted 10 times and slowly add it to 990 μL of blank serum to obtain the S1 standard curve solution.

[0129] The working solution was first further diluted before being used to prepare the standard curve solution with blank serum. This was mainly to further reduce the acetonitrile ratio and avoid slight protein precipitation in the porcine blank serum after subsequent treatments, which would affect the low concentration values. The standard curve working solution was not directly diluted to the desired concentration because a higher concentration is more conducive to preservation.

[0130] Table 5. Theoretical Concentration of Standard Curve Solution

[0131] 2. Protein internal standard precipitant

[0132] 2.1 Preparation of internal standard stock solution

[0133] Fifteen isotopic internal standards for bile acids and three bile acid precursors were taken and diluted with acetonitrile according to Table 6 to obtain internal standard stock solutions. The concentrations of each internal standard in the internal standard stock solutions are shown in Table 6.

[0134] Table 6. Preparation of Internal Standard Stock Solution

[0135] 2.2 Preparation of protein internal standard precipitant

[0136] Prepare the internal standard stock solution by adding 1.5 mL of acetonitrile to 0.1 mL of the internal standard stock solution, mix well, and store at -20°C protected from light.

[0137] 3. Quality control products

[0138] 3.1 Preparation of working solution for quality control products

[0139] Using the mixed intermediate solution prepared in section 1.2 as the mother solution (solution A), and a 30% acetonitrile aqueous solution as the diluent, the mixed intermediate solution was diluted according to the volumes shown in Table 7 to prepare two quality control working solutions, QL and QH. The theoretical concentrations of each standard in the QL and QH quality control working solutions are shown in Table 8.

[0140] Table 7. Preparation of Working Solution for Quality Control Products

[0141] Table 8. Theoretical concentrations of various standards in the working solution of quality control materials.

[0142] 3.2 Preparation of quality control products

[0143] a. Blank serum matrix: Clinical residual heparin anticoagulated normal human blank serum was used. After repeated freeze-thaw cycles 5 times, activated carbon was added to prepare a serum suspension with an activated carbon concentration of 0.2 g / mL. The suspension was vortexed at room temperature for 2 hours to ensure sufficient contact and adsorption. The suspension was then centrifuged at 13000 rpm and 4℃ for 20 minutes. The supernatant was collected and filtered to obtain the blank human serum required for this experiment.

[0144] b. Preparation of Quality Control Samples: Take the quality control working solutions (QL, QH) prepared in 3.1 and mix them with blank human serum at a ratio of 1:99 (V:V) to prepare quality control samples QL and QH. The theoretical concentrations of each standard in quality control samples QL and QH are shown in Table 9. Taking QL as an example: Take 10 μL of QL quality control working solution and slowly add 990 μL of blank human serum to obtain quality control sample QL.

[0145] Table 9. Theoretical Concentration of Each Standard in the Quality Control Materials

[0146] Example 2

[0147] The standard curve solution and serum samples were tested using the following method:

[0148] 1. Protein precipitation: Accurately pipette 50 μL of standard curve solution / serum sample into a centrifuge tube, add 160 μL of protein internal standard precipitant, vortex to mix for 30 seconds, and then centrifuge at 13000 RPM for 10 minutes.

[0149] 2. Dilution: Take 100 μL of supernatant and transfer it to a 96-well deep plate. Add 100 μL of deionized water to dilute it 1:1. Mix well using a shaker and centrifuge for 1 min.

[0150] 3. Detection on the instrument: Take 5 μL of the supernatant after centrifugation and perform LC-MS detection. The liquid chromatography and mass spectrometry conditions are shown below.

[0151] Instrument: AB SCIEX Triple Quad TM 4500MD Liquid Chromatography-Tandem Mass Spectrometry Detection System

[0152] (1) Liquid chromatography conditions

[0153] Chromatographic column: Waters UPLC BEH C18 1.7um; 2.1*100mm

[0154] Mobile phase A: 0.01% formic acid-water solution

[0155] Mobile phase B: Acetonitrile solution

[0156] Flow rate: 0.3 mL / min

[0157] Column temperature: 45℃

[0158] Injection volume: 5 μL

[0159] Elution gradient:

[0160] Table 10 Elution Gradient Table

[0161] (2) Mass spectrometry conditions

[0162] Ion source parameters:

[0163] Acquisition mode: ESI+ / -

[0164] Table 11 Data Acquisition Parameter Table

[0165] Ion pair parameters:

[0166] Table 12 Ion Pair Parameters Note: Mass spectrometry parameters need to be adjusted according to the specific instrument.

[0167] 4. Draw the standard curve

[0168] The standard curve is plotted and the standard curve equation is fitted based on the test results of the standard curve solution.

[0169] (1) Plot the standard curve fitting graph: Plot the standard curve with the concentration of the 6 standard curve solutions (S1~S6) as the abscissa (x) and the ratio of the actual detection peak area of ​​the 6 standard curve solutions to the peak area of ​​their respective internal standards as the ordinate (y).

[0170] (2) Fitting the standard curve equation: Linear regression was performed on the concentration (x) using the peak area ratio (y) of the six standard curve solutions. The regression equation was obtained as: y = a + bx, where y is the ordinate, x is the abscissa, a is the intercept, and b is the slope. The correlation coefficient (r) was calculated, and r should not be less than 0.990. The results are shown in Table 13.

[0171] Table 13 Linear Regression Curves

[0172] The linearity of the standard curve is r≥0.995, indicating a good linear relationship.

[0173] 5. Calculate sample concentration

[0174] Calculation of sample test results: Based on the test results of serum samples, the ratio of the peak area of ​​15 bile acids and 3 bile acid precursors in the sample to the peak area of ​​their internal standard is substituted into the above standard curve equation to calculate the concentration of 15 bile acids and 3 bile acid precursors in the sample.

[0175] Clinical serum samples containing 15 bile acid profiles and 3 bile acid precursors were tested according to the aforementioned method. The total ion chromatogram of the 15 bile acid profiles and 3 bile acid precursors obtained by mass spectrometry is shown in Figure 1.

[0176] As shown in Figure 1, the method of the present invention can effectively separate 15 bile acid profiles and 3 bile acid precursors to meet the requirements for quantitative detection.

[0177] Furthermore, based on the linear regression fitting curves shown in Figure 1 and Table 13, the concentrations of 15 bile acids and 3 bile acid precursors in clinical serum samples were calculated, as shown in Table 14:

[0178] Table 14. Concentration Calculation Table of 15 Bile Acid Profiles and 3 Bile Acid Precursors

[0179] Experimental Example 1

[0180] This experiment investigated different mobile phase compositions while keeping all other steps and conditions constant. Except for the mobile phase composition, all other steps and conditions were performed as in Example 2. The results are shown in Table 15.

[0181] Table 15 Comparison of peak elution characteristics for different mobile phases and additives

[0182] The results showed that when mobile phase A was 0.01% formic acid-water solution and mobile phase B was acetonitrile, it was possible to simultaneously detect 15 bile acid profiles and distinguish isomers of 3α,7α-dihydroxycobalamin prostaglandin (DHCA) and 3α,7α,12α-trihydroxycholestannic acid (THCA), thereby simultaneously detecting DHCA, THCA and C4 (7α-hydroxy-4-cholesterol-3-one).

[0183] Experimental Example 2

[0184] This experiment investigated the methods for selecting and preparing blank plasma. With all other steps and conditions constant, only the type and processing method of blank plasma were adjusted.

[0185] Except for the method of preparing the blank serum matrix used in preparing the standard curve solution, all other steps and conditions were performed in accordance with Example 2. The serum samples were first pretreated with an internal standard containing acetonitrile, a protein precipitant, and then the background peak areas of 15 bile acids and 3 bile acid precursors were detected by tandem mass spectrometry.

[0186] 1. The results of the tests using untreated heparin-anticoagulated normal swine serum, heparin-anticoagulated normal bovine serum, and heparin-anticoagulated normal sheep serum are shown in Table 16 below:

[0187] Table 16 Background Results of Serums from Different Sources

[0188] Based on the results in the table above, using heparin-anticoagulated normal swine serum as a blank matrix yielded the best results.

[0189] 2. The following comparisons were made using untreated heparin-anticoagulated normal porcine serum, serum adsorbed with activated charcoal using a simple suspension method (0.05 g / mL), serum adsorbed with activated charcoal using a simple suspension method (0.1 g / mL), serum adsorbed with activated charcoal using a simple suspension method (0.2 g / mL), serum adsorbed with activated charcoal using a suspension method after two freeze-thaw cycles (0.2 g / mL), serum adsorbed with activated charcoal using a suspension method after five freeze-thaw cycles (0.2 g / mL), and serum adsorbed with activated charcoal using a suspension method after ten freeze-thaw cycles (0.2 g / mL). The test results are shown in Table 17 below.

[0190] Table 17 shows the test results of different methods used to treat the blank matrix.

[0191] Based on the table above, the best results were obtained by using heparin to anticoagulate normal porcine serum, repeatedly freezing and thawing it five times, and then adding activated charcoal to prepare a serum suspension with an activated charcoal concentration of 0.05–0.2 g / mL.

[0192] This invention utilizes repeated freeze-thaw temperature changes to destroy endogenous active substances and sedimented impurities in the blood, and combines this with suspension adsorption technology for effective adsorption, effectively preparing a matrix blank serum background that approximates the type of test sample, thus effectively reducing background interference.

[0193] Example 1 - Linearity Verification

[0194] 1. Linearity Validation Requirements: Take clinical linearity validation samples from LV1 to LV5 and perform linearity validation according to YY / T1789.4-2022. The relative deviation of the five concentration results from the theoretical values ​​should be within ±15%, R0. 2 ≥0.98.

[0195] 2. Detection methods and results: Clinical samples of LV1 and LV5 with concentrations close to those of the standard curve S1 and S6 were collected and mixed in volume ratios of 8:2, 5:5, and 2:8 respectively to obtain mixed clinical samples of LV2, LV3, and LV4, which were collectively referred to as linear validation samples LV1 to LV5.

[0196] The linear verification samples of LV1 to LV5 were tested according to the method in Example 2, and the concentration of each substance in the linear verification samples of LV1 to LV5 was quantitatively calculated according to the standard curve shown in Table 13. The test was repeated 3 times to obtain the mean value and the relative deviation from the target value (theoretical concentration) was calculated.

[0197] The results of linear verification and the results of relative deviation are recorded in Table 18.

[0198] Table 18. Results of linear validation and relative bias for clinical linear validation samples from LV1 to LV5.

[0199] The test results show that the relative deviation of the test results for the clinical linear validation samples is generally within 7%, R 2 All are greater than 0.98, satisfying the requirement that the relative deviation is within ±15%, R 2 Linearity verification requirement of ≥0.98.

[0200] Example 2 - Quantitative Lower / Upper Limit Detection

[0201] 1. Acceptance criteria: The bias of the quantitative / upper limit sample test results is within 85%-115%.

[0202] 2. Experimental Procedure: Collect 6 clinical samples each of low-concentration LLOQ and high-concentration ULOQ. The low-concentration clinical samples are close to "S1", and the high-concentration clinical samples are close to "S6". Detect LLOQ (1-6) and ULOQ (1-6) samples according to the method in Example 2. Quantitatively calculate the concentration of each substance in LV1-LV5 samples based on the standard curve shown in Table 13. Repeat the detection 3 times to obtain the mean value and calculate the bias from the target value (theoretical concentration).

[0203] The specific testing methods are as follows:

[0204] (1) Protein precipitation: Accurately aspirate 50 μL of the above clinical sample into a centrifuge tube, add 160 μL of protein internal standard precipitant, shake to mix for 30 seconds, and then centrifuge at 13000 RPM for 10 minutes.

[0205] (2) Dilution: Take 100 μL of supernatant and transfer it to a 96-well deep plate. Add 100 μL of deionized water to dilute it 1:1. Mix well using a shaker and centrifuge for 1 min.

[0206] (3) LC-MS detection: After centrifugation, 5 μL of supernatant was taken and detected by LC-MS. The LLOQ (1-6) and ULOQ (1-6) samples were quantitatively analyzed using the standard curve to obtain the detection value.

[0207] 3. Experimental Results:

[0208] Table 18. Detection values ​​and bias results for 6 LLOQ and ULOQ samples.

[0209] 4. Conclusion: The results show that the sample detection bias of the lower limit of quantitation of 15 bile acid profiles and 3 bile acid precursors in serum samples is between 85% and 115%, which meets the requirements.

[0210] The spectra of 15 bile acids and the quantitative detection range of 3 bile acid precursors are shown in Table 19 and Figures 2-35.

[0211] Table 19. Spectra of 15 bile acids and quantitative detection ranges of 3 bile acid precursors, along with corresponding appendices.

[0212] Example 3 – Accuracy (Recovery Rate)

[0213] 1. Acceptance criteria: The average recovery rate for each concentration sample is between 85% and 115%.

[0214] 2. Experimental methods: Six clinical serum samples from different sources were collected, mixed, and then mixed with low and high concentration standard solutions (concentrations are shown in "Table 20") to prepare "QBL" and "QBH". Three batches of each sample were tested, and five replicates were performed for each concentration sample. The average recovery rate was calculated, and the results are shown in Table 20.

[0215] 3. Experimental data and results:

[0216] Table 20 Calculation of Spike Recovery Rate in Clinical Serum Samples

[0217] 4. Conclusion: The results show that the accuracy of the 18 bile acid spectra is between 89.8% and 99.6%, therefore the accuracy of this method meets the requirements.

[0218] Example 4 – Intra-batch & Inter-batch Precision

[0219] 1. Acceptance criteria: Intra-batch precision (CV) ≤ 15%

[0220] 2. Experimental methods:

[0221] Following the preparation method of the quality control material in Example 1, human serum from different sources was collected. Following the method of processing heparin-anticoagulated normal pig blank serum into blank serum matrix in Example 1.4, it was processed into mixed blank serum matrix. Low and high concentrations of quality control material working solution were added to prepare "QL" and "QH" respectively. Three batches of each sample were measured, and five replicates were performed for each concentration sample. CV was calculated, and the detection calculation results are shown in "Table 21".

[0222] 3. Experimental Results:

[0223] Table 21 Results of Intra-batch and Inter-batch Detection Coefficients of Variation for Low and High Concentration Quality Controls

[0224] 4. Conclusion:

[0225] The results show that the intra-batch precision CV of the 18 bile acid spectra is between 1% and 12%, which meets the requirement that the intra-batch precision CV is not greater than 15%. Therefore, the intra-batch precision of this method meets the requirements.

[0226] The results show that the inter-batch precision CV of the 18 bile acid spectra is between 3% and 11%, which meets the requirement that the inter-batch precision CV is not greater than 15%. Therefore, the inter-batch precision of this method meets the requirements.

[0227] Example 5 – Matrix Effect

[0228] 1. Acceptance criteria: Relative deviation less than ±20%;

[0229] 3. Experimental methods:

[0230] Mixed serum matrix sample A: A sample collected from six different clinical samples and then mixed together;

[0231] Solution matrix sample B1: A low-concentration standard solution prepared using 30% acetonitrile aqueous solution as the matrix, according to the preparation method of the standard curve solution;

[0232] Solution matrix sample B2: A high-concentration standard solution prepared using 30% acetonitrile aqueous solution as the matrix, according to the preparation method of the standard curve solution;

[0233] The relative matrix effect was evaluated using a mixed experimental method. Mixed serum matrix sample A, and solution matrix samples B1 and B2 were measured; mixtures of A with B1 and B2 in a 1:1 (V:V) ratio were measured three times in parallel. The matrix effect of the proposed detection method was evaluated by pretreatment of the compounds in these three matrix samples and mass spectrometry detection. The results are shown in Table 22.

[0234] 3. Experimental Results:

[0235] Table 22 Results of the mixed experimental method for evaluating matrix effect

[0236] 4. Conclusion: The relative deviation of the 18 relative matrix effect tests of serum bile acids was -10% to 10%, which meets the detection requirement of relative deviation less than ±20%.

[0237] In summary, the above-mentioned effect examples, based on the relevant guidelines such as "Development and Validation of Clinical Detection Methods for Liquid Chromatography-Tandem Mass Spectrometry" and "CNAS-GL037 Performance Validation Guidelines for Quantitative Clinical Chemistry Testing Procedures", have validated the performance of the method for detecting 18 bile acids in this application. A total of 9 items were validated, namely matrix effect, detection limits, linearity evaluation, accuracy, and precision validation, all of which meet the requirements. Furthermore, it can simultaneously detect 15 bile acid profiles and 3 bile acid precursors (cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, 3α,7α-dihydroxycobalamin prostaglandin, 3α,7α,12α-trihydroxycholestannic acid, and 7α-hydroxy-4-cholesterol-3-one), which to some extent addresses the clinical need to assess the enterohepatic cycle, which consists of the biliary system, intestines, portal circulation, and hepatocytes.

[0238] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A kit for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum, characterized in that, The 15 bile acid profiles include: cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, and taurocholic acid. The three bile acid precursors include: 3α,7α-dihydroxycobalt prostatic acid, 3α,7α,12α-trihydroxycholestan acid, and 7α-hydroxy-4-cholesterol-3-one; The kit includes: standard curve solution, protein internal standard precipitant, quality control materials, and mobile phase; The protein internal standard precipitant includes an internal standard stock solution, which is a mixed solution containing isotopic internal standards of the 15 bile acid profiles and 3 bile acid precursors; The standard curve solution was prepared using a blank serum matrix and a standard curve working solution, and the standard curve working solution included a mixed solution of the 15 bile acid spectra and 3 bile acid precursors. The quality control sample was prepared using a blank serum matrix and a quality control working solution, wherein the quality control working solution comprised a mixed solution of the 15 bile acid profiles and 3 bile acid precursors. The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.01-0.1% aqueous solution of formic acid and mobile phase B is an acetonitrile solution.

2. The reagent kit according to claim 1, characterized in that, The protein internal standard precipitant is prepared by internal standard stock solution and acetonitrile, wherein the volume ratio of internal standard stock solution to acetonitrile is 1:15 to 1:

20.

3. The reagent kit according to claim 1, characterized in that, The isotopic internal standards for the 15 bile acid profiles include cholic acid-d4, deoxycholic acid-d4, chenodeoxycholic acid-d4, ursodeoxycholic acid-d4, lithocholic acid-d4, glycocholic acid-d4, glycodeoxycholic acid-d4, glycochenodeoxycholic acid-d4, glycoursodeoxycholic acid-d4, glycolithocholic acid-d4, taurocholic acid-d4, taurodeoxycholic acid-d4, taurochenodeoxycholic acid-d4, and tauroursodeoxycholic acid-d4. The isotopic internal standards for the three bile acid precursors include 3α,7α-dihydroxycobalt prostaglandin-d3, 3α,7α,12α-trihydroxycholestannic acid-d3, and 7α-hydroxy-4-cholesterol-3-one-d7.

4. The reagent kit according to claim 1, characterized in that, The blank serum matrix is ​​prepared by using heparin-anticoagulated normal blank serum, repeatedly freezing and thawing 5 to 10 times, adding activated carbon to prepare a serum suspension, vortexing to allow it to fully adsorb, centrifuging, and filtering the supernatant to obtain the blank serum matrix.

5. The reagent kit according to claim 4, characterized in that, The concentration of activated charcoal in the serum suspension is 0.05–0.2 g / mL; And / or, the temperature of the vortex is 20–30°C, and the time is 1–3 hours; And / or, the centrifugation speed is 12000-13000 RPM, the temperature is 3-6℃, and the time is 5-20 min.

6. The reagent kit according to claim 1, characterized in that, The blank serum matrix used in preparing the standard curve solution was prepared from heparin-anticoagulated normal porcine blank serum; And / or, the blank serum matrix used in preparing the quality control product is prepared from heparin-anticoagulated normal human blank serum.

7. A method for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum, using the kit described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Take a serum sample, add protein internal standard precipitant, and shake to mix; (2) After centrifugation, the supernatant was taken, diluted, and then detected by liquid chromatography-tandem mass spectrometry. (3) Calculate the concentrations of 15 bile acids and 3 bile acid precursors in serum samples based on the standard curve.

8. The method according to claim 7, characterized in that, The chromatographic column used for the instrumental detection in step (2) is a Waters UPLC BEH C18 column; the column temperature is 40-45℃ and the flow rate is 0.28-0.33mL / min.

9. The method according to claim 7, characterized in that, The method for plotting the standard curve in step (3) is as follows: take the standard curve solution, add the protein internal standard precipitant, shake to mix, centrifuge to take the supernatant, dilute and then test it on the instrument. Plot the standard curve with the concentration of the standard curve solution as the abscissa and the ratio of the peak area to the internal standard peak area as the ordinate.

10. The method according to claim 7, characterized in that, During the on-machine testing in step (2), the gradient elution program is as follows:

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