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Improved method for detecting 15 bile acids in human serum by high performance liquid chromatography-tandem mass spectrometry

A technology of high performance liquid chromatography and tandem mass spectrometry, which is applied in the field of detection of 15 kinds of bile acids in human serum by improved high performance liquid chromatography and tandem mass spectrometry, which can solve the problem of inability to accurately distinguish different types of bile acids, low sensitivity, and unsatisfactory sensitivity Clinical testing needs and other issues

Inactive Publication Date: 2020-12-18
杭州凯莱谱精准医疗检测技术有限公司
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Problems solved by technology

[0003] At present, the commonly used bile acid detection methods mainly include enzyme cycling method and high performance liquid chromatography. These detection methods mainly have the following problems: the enzyme cycling method can only detect the content of total bile More accurate analysis, diagnosis and identification of diseases has limited clinical guidance; high-performance liquid chromatography requires derivatization, especially taurine-bound bile acids need to be hydrolyzed and then derivatized, and the pre-treatment steps are very complicated; in addition , the ultraviolet absorption of bile acids is weak, and the sensitivity of the ultraviolet detector used in chromatography cannot meet the needs of clinical detection, so the clinical application of this method has great limitations
In short, the existing traditional methods for the detection of bile acids not only have cumbersome pretreatment methods, require a long manual operation process, take a long time for subsequent instrument detection, limit the types of bile acid detection, but also have poor detection specificity and low sensitivity. Clinical application of bile acid detection method

Method used

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  • Improved method for detecting 15 bile acids in human serum by high performance liquid chromatography-tandem mass spectrometry
  • Improved method for detecting 15 bile acids in human serum by high performance liquid chromatography-tandem mass spectrometry
  • Improved method for detecting 15 bile acids in human serum by high performance liquid chromatography-tandem mass spectrometry

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Effect test

Embodiment 1

[0057] Example 1 An improved high performance liquid chromatography tandem mass spectrometry detection method for 15 bile acids in human serum

[0058] 1. Solution preparation

[0059] Preparation of primary stock solution: Accurately weigh CA, DCA, CDCA, LCA, GCA, GCDCA, GUDCA, GLCA, TCA, TDCA, TCDCA, TUDCA and TLCA standards, add 20% methanol solution to dissolve and dilute to obtain a concentration of 2mg / mL of primary stock solution; accurately weigh 20 mg of UDCA and GDCA standard substances, add 20% methanol solution to dissolve and dilute respectively to obtain a primary stock solution with a concentration of 4 mg / mL;

[0060] Preparation of secondary mixed stock solution (MIX01): Add appropriate amount of 15 primary stock solutions to the same centrifuge tube in turn, vortex and mix well to obtain secondary mixed stock solution (MIX01), the specific concentration of which is shown in Table 4 Show.

[0061] Table 4 Preparation of the secondary mixed stock solution of...

Embodiment 2

[0090] Embodiment 2: A detection kit for detecting 15 kinds of bile acids

[0091] The composition of the kit is shown in Table 10:

[0092] Table 10 Kit Composition

[0093]

[0094] Different precipitation agents and different diluent formulations are shown in Table 11 and Table 12 respectively:

[0095] Table 11 different precipitant formulations

[0096]

[0097] Table 12 Different Diluent Formulations

[0098]

[0099]

[0100] The bile acid internal standard solution includes 200ng / mL deuterated glycocholic acid (GCA-d5), 100ng / mL deuterated taurocholic acid (TCA-d5), 50ng / mL deuterated taurodeoxycholic acid ( TDCA-d4), deuterated tauroursodeoxycholic acid (TUDCA-d5) at 500 ng / mL.

[0101] The maximum concentration of the standard curve of 15 kinds of bile acids is from 500-6000ng / mL, and then serially diluted to obtain a series of standard curve solutions corresponding to 15 kinds of analytes. The quality control samples of bile acids include low quality...

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Abstract

The invention belongs to the technical field of biology, and relates to an improved high performance liquid chromatography-tandem mass spectrometry detection method for 15 bile acids in human serum. The method comprises the steps of protein precipitation, sample dilution and analysis and detection by high performance liquid chromatography-tandem mass spectrometry. The method has the advantages ofhigh efficiency, low cost, simple operation, high accuracy and sensitivity and the like, and is suitable for rapid detection of a large number of clinical samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an improved high performance liquid chromatography tandem mass spectrometry detection method for 15 kinds of bile acids in human serum. The steps of the method include protein precipitation, sample dilution, and high performance liquid chromatography tandem mass spectrometry for analysis and detection , has the advantages of high efficiency, low cost, easy operation, high accuracy and sensitivity, and is suitable for rapid detection of a large number of clinical samples. Background technique [0002] Bile acid is the main component of bile and is a general term for a class of cholic acid. Bile acids have a variety of isomers, which can be divided into free and conjugated bile acids according to their structures. Free bile acids mainly include cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid, and conjugated bile acids It is the combination product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72
CPCG01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N2030/324
Inventor 马金飞杨青青孔子清刘华芬
Owner 杭州凯莱谱精准医疗检测技术有限公司
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