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CN-122017068-A - Bile acid high-flux detection method based on ultra-high performance liquid chromatography tandem mass spectrometry and application thereof

CN122017068ACN 122017068 ACN122017068 ACN 122017068ACN-122017068-A

Abstract

The invention provides a bile acid high-flux detection method based on ultra-high performance liquid chromatography tandem mass spectrometry and application thereof. The method comprises the steps of pretreating a sample to be detected, collecting an eluent through a C18 solid-phase microextraction column to serve as a sample solution, separating and detecting by adopting an ultra-high performance liquid chromatography tandem mass spectrum, wherein a mobile phase adopted by the ultra-high performance liquid chromatography comprises a mobile phase A, a mobile phase B, and a gradient elution, wherein the mobile phase A comprises a 0.095-0.105% formic acid aqueous solution containing 4.95-5.05mM ammonium acetate, the mobile phase B comprises an acetonitrile-methanol solution with the volume ratio of 3 (0.95-1.05), and a linear regression equation of each bile acid subtype is established by mixing standards with different concentrations for quantitative calculation. The invention establishes a high-efficiency extraction and detection method for bile acid samples, realizes separation and accurate quantification of 50 bile acid compounds, and obviously reduces the false positive problem caused by coelute of isomer bile acid in the traditional detection method.

Inventors

  • CAO YICHEN
  • Meng Qilu
  • LI TIANYUN

Assignees

  • 青岛百谱生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260205

Claims (10)

  1. 1. A bile acid high-flux detection method based on ultra-high performance liquid chromatography tandem mass spectrometry is characterized by comprising the following steps: (1) Pretreating a sample to be detected, passing through a C18 solid-phase microextraction column, and collecting eluent as a solution of the sample to be detected; (2) Separating and detecting a sample solution and a standard mixed standard solution by adopting ultra-high performance liquid chromatography tandem mass spectrometry, wherein a mobile phase adopted by the ultra-high performance liquid chromatography comprises a mobile phase A, a mobile phase B, a gradient elution and a gradient elution program, wherein the mobile phase A comprises 0.095-0.105% formic acid aqueous solution containing 4.95-5.05 mM ammonium acetate, the mobile phase B comprises acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 3 (0.95-1.05): 0 th to 0.5 min th, the volume fraction of the mobile phase A is 90 to 90.5%, and the balance is the mobile phase B; 0.5-1.5 min, the volume fraction of the mobile phase A is changed from 90-90.5% to 78-78.5% at uniform speed, and the rest is the mobile phase B; 1.5-2.5 min, the volume fraction of the mobile phase A is changed from 78-78.5% to 68-68.5% at uniform speed, and the rest is the mobile phase B; 2.5-3.5 min, the volume fraction of mobile phase A is 68-68.5%, and the rest is mobile phase B; 3.5-5.5 min, the volume fraction of the mobile phase A is changed from 68-68.5% to 64.5-65% at uniform speed, and the rest is the mobile phase B; 5.5-9 min, the volume fraction of the mobile phase A is changed from 64.5-65% to 58-58.5% at uniform speed, and the rest is the mobile phase B; 9-10 min, the volume fraction of the mobile phase A is changed from 58-58.5% to 57-57.5% uniformly, and the rest is the mobile phase B; From 10 th to 18 th min th, the volume fraction of mobile phase A is changed from 57-57.5% to 33.5% at uniform speed, and the rest is mobile phase B; 18-19.2 min, the volume fraction of the mobile phase A is changed from 33-33.5% to 5-5.5% at uniform speed, and the rest is the mobile phase B; 19.2-20.3 min, the volume fraction of mobile phase A is 5-5.5%, and the rest is mobile phase B; The volume fraction of the mobile phase A is changed from 5-5.5% to 90-90.5% from 20.3-20.4 min%, and the rest is the mobile phase B; 20.4-23 min, the volume fraction of the mobile phase A is 90-90.5%, and the rest is the mobile phase B; (3) A linear regression equation of each bile acid subtype compound is established through standard substance mixing marks with different concentrations and is used for quantitative calculation.
  2. 2. The method for detecting bile acid with high flux based on ultra performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the step (1), the step of preprocessing the sample to be detected comprises: Mixing the collected sample to be tested with the extracting solution, shaking after freezing with dry ice to promote sample cracking, carrying out ice water bath ultrasonic extraction, centrifuging after freezing with dry ice to collect supernatant, filtering, collecting filtrate, mixing the filtrate with water, passing through a C18 solid-phase microextraction column, eluting with pure water, eluting with pure methanol for at least 3 times, and collecting the eluent as a sample solution.
  3. 3. The high-throughput detection method of bile acid based on ultra-high performance liquid chromatography tandem mass spectrometry according to claim 2, wherein the extracting solution is acetonitrile-methanol-water solution, wherein the volume ratio of acetonitrile, methanol and water is (1.9-2.1): 0.9-1.1; preferably, the dry ice is frozen for a period of 4-6 min; preferably, the condition of the oscillation is that 1200-1250 rpm oscillates 10-15 min; Preferably, the time of the ultrasonic extraction step is 8-12 min; Preferably, the conditions of the centrifugation step are that the rotation speed is 11000-12000 rpm, and the centrifugation is carried out at 0-4 ℃ for 8-12 min; Preferably, the filtering uses a disposable needle filter, and the filter membrane is made of nylon 66 with the thickness of 0.22 mu m.
  4. 4. The method for detecting bile acid with high flux based on ultra-high performance liquid chromatography tandem mass spectrometry according to claim 2 or 3, wherein the volume ratio of the filtrate to water is 1 (3.5-4.5); Preferably, the packing of the C18 solid phase microextraction column is Sep-Pak C18 1 cc Vac Cartridge.
  5. 5. The method for high throughput detection of bile acid based on tandem mass spectrometry of any one of claims 1 to 4, wherein in step (2), the column of the ultra performance liquid chromatography is Waters ACQUITY UPLC BEH C columns, model number is 1.7 μm,2.1 mm ×100 mm, and column temperature is 34 to 36 ℃.
  6. 6. The method for high throughput detection of bile acid based on tandem mass spectrometry of any one of claims 1 to 5, wherein in step (2), the model of ultra-high performance liquid chromatography is ACQUITY UPLC I-Class PLUS, waters, and the sample injection amount is 1.8-2.2 μl.
  7. 7. The method for detecting bile acid with high flux based on ultra performance liquid chromatography tandem mass spectrometry according to any one of claims 1 to 6, wherein in the step (2), the mass spectrometry condition is that electrospray ESI ion source is adopted, negative ion mode detection is adopted, instrument parameters are that gas curtain gas is 35 psi, ion spray voltage is-4500V, ion source temperature is 550 ℃, atomizing gas is 50 psi, and auxiliary gas is 55 psi.
  8. 8. The method for high throughput detection of bile acid based on ultra performance liquid chromatography tandem mass spectrometry according to any one of claims 1 to 7, wherein in step (2), the standard mixed labeling solution comprises: dehydrolithocholic acid, isophthalic acid, lithocholic acid, 23-demethdeoxycholic acid, 7-ketolithocholic acid, 12-ketolithocholic acid, procholic acid, ursodeoxycholic acid, hyodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, isodeoxycholic acid, dehydrocholic acid, 7, 12-diketo lithocholic acid, 7-ketodeoxycholic acid, 12-dehydrocholic acid, 3-dehydrocholic acid, ursolic acid, alpha-murine cholic acid, beta-murine cholic acid, hyodeoxycholic acid, allopcholic acid, cholic acid, glycolithocholic acid, glycoursodeoxycholic acid, glycohyodeoxycholic acid, glycodeoxycholic acid, glycodehydrocholic acid, glycocholic acid, taurocholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, tauro-alpha-murine cholic acid, taurocholic acid, iso Bie Dan cholic acid, murine deoxycholic acid, iso-ursodeoxycholic acid, iso-hyodeoxycholic acid, 3 beta-deoxycholic acid, norcholic acid, 3 beta-cholic acid, omega-murine cholic acid, glycochenodeoxycholic acid, lithocholic acid sulfuric acid, glycohyodeoxycholic acid, tauro-beta-murine cholic acid, taurocholic acid, and chenodeoxycholic acid-3 beta-glucuronide.
  9. 9. The method for high-throughput detection of bile acid based on ultra-high performance liquid chromatography tandem mass spectrometry of claim 8, wherein the concentration range of each bile acid subtype standard in the standard mixed labeling solution is 0.1-5000 ng/mL.
  10. 10. Use of the bile acid high-throughput detection method based on ultra-high performance liquid chromatography tandem mass spectrometry according to any one of claims 1-9 in bile acid detection.

Description

Bile acid high-flux detection method based on ultra-high performance liquid chromatography tandem mass spectrometry and application thereof Technical Field The invention belongs to the field of analysis and detection, and particularly relates to a bile acid high-flux detection method based on ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof. Background The bile acid is used as a core product of cholesterol metabolism, is not only a key medium for emulsifying fat, regulating fat and fat-soluble vitamins, transporting and distributing, but also a signal molecule participating in glycolipid metabolism regulation through FXR, TGR5 and other receptor channels, and has important physiological functions. Bile acid is used as main component of bile, its subtype composition and dynamic balance are directly related to liver and intestine circulation function, and specific change is shown in pathological processes such as non-alcoholic fatty liver, cholestasis of gestation (ICP) and inflammatory bowel disease. In the medical research direction, the accurate qualitative and quantitative analysis of bile acid substances has practical significance for related disease research, liver function evaluation and related drug metabolism and treatment effect examination. Accurate detection of bile acid components is therefore of great importance in medical research and disease diagnosis in the liver and digestive metabolic directions. Bile acids can be structurally classified into free and bound types. Free bile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. The conjugated bile acid is the conjugate product of free bile acid and glycine or taurine, and mainly comprises glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid and the like. Bile acids can be classified into primary bile acids, secondary bile acids and tertiary bile acids according to their source. Bile acids synthesized directly from cholesterol in hepatocytes are referred to as primary bile acids, and include cholic acid, chenodeoxycholic acid, and their binding products with glycine or taurine. The primary bile acid is acted by bacteria in intestinal tract, and the bile acid generated after 7 alpha-hydroxy deoxidation is called secondary bile acid, and the secondary bile acid mainly comprises deoxycholic acid and lithocholic acid and the combination product generated by combining the deoxycholic acid and glycine or taurine. The tertiary bile acid is the metabolite of reabsorption secondary bile acid in liver and intestinal tract, including sulfocholic acid and ursodeoxycholic acid, etc. Due to limitations in detection technology, current clinical detection is focused on total bile acid or limited species bile acid analysis (typically < 30). The problem of poor quantitative qualitative reliability exists in the detection of bile acid by a kit enzyme circulation method and a high performance liquid chromatography method, meanwhile, more complex steps such as derivatization are involved in the pretreatment process of the sample, the requirements on the reaction temperature and the derivatization reagent are higher, the detection flux is difficult to promote, the sample reproducibility is relatively poor, the experimental cost is high, and the two detection means have larger limitations in the clinical application process and the like. Meanwhile, the samples involved in bile acid detection are often biological samples such as blood plasma, intestinal contents, liver tissues and the like, the matrix of the samples is complex, and the extracting solution obtained after the conventional extraction method is used for processing contains inorganic salt and other impurities, so that the baseline noise of the sample detection, the response value of the compounds and the stability of the method are adversely affected, and the chromatographic column and the ion source needle are greatly damaged. The solid-phase microextraction column with the C18 filler is used for purifying the sample extracting solution, so that the purifying method is stable and efficient, impurities such as hydrophilic salt and the like can be effectively removed in a good recovery rate of substances with weaker polarities, and the solid-phase microextraction column is matched with a matched solid-phase microextraction device, so that high-flux sample treatment can be realized, compared with the traditional method, the method has the advantages that the treatment time is not additionally increased, the structure of the compound is not changed, the data quality is improved, the experimental efficiency is ensured, the timeliness is ensured for mass sample extraction, and the degradation of bile acid substances in the pretreatment process can be avoided. The ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method has the advantages of high qualitative ac