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Method for simultaneously detecting five anti-tuberculosis drugs in blood plasma via UPLC-MS/MS method

An anti-tuberculosis and plasma technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that cannot fully meet the needs of clinical measurement, long analysis time, complicated pre-treatment, etc., and achieve short processing time, short analysis time, and pre-processing Handle simple effects

Active Publication Date: 2019-08-20
MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently, ultra-high performance liquid chromatography-tandem mass spectrometry is the main method for the detection of anti-tuberculosis drugs. Meeting Clinical Measurement Needs

Method used

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  • Method for simultaneously detecting five anti-tuberculosis drugs in blood plasma via UPLC-MS/MS method
  • Method for simultaneously detecting five anti-tuberculosis drugs in blood plasma via UPLC-MS/MS method
  • Method for simultaneously detecting five anti-tuberculosis drugs in blood plasma via UPLC-MS/MS method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Different chromatographic columns, mobile phase ratios and gradient elution methods were used to analyze the detected standard substances.

[0055] (1) The chromatographic column Shim-pack XR-ODSⅢ (2.0mm i.d×50mm, 1.6μm); the pre-column Shim-pack GIST-HP(G) (2μm C18, 2.1×10mm) was used to separate and analyze the standard. The mobile phase AB adopts different proportions and gradient elution methods. The peaks of isoniazid and ethambutol in the analyte both elute at about 0.5 min, and the above two analytes are not retained on this chromatographic column. Wherein the chromatographic mobile phase is set as follows: A (water phase): 0.01% (w / w) formic acid aqueous solution, B (organic phase): acetonitrile solution with formic acid concentration of 0.01% (w / w).

[0056]

[0057] The chromatographic peaks of the five standard substances measured are as follows: figure 1 ,Depend on figure 1 It can be seen that ethambutol and isoniazid are almost not retained, and the pe...

Embodiment 2

[0076] Three plasma samples from patients infected with Mycobacterium tuberculosis were collected respectively, named sample 1, sample 2, and sample 3.

[0077] (1) a. Standard product handling:

[0078] Take 90 μL of blank plasma, add 10 μL of mixed standard solutions of different concentrations, vortex and mix, add 10 μL of mixed isotope internal standard working solution, then add 300 μL of acetonitrile, vortex for 1 min, and centrifuge at 12000 r / min for 5 min; absorb 300 μL of supernatant in another Add 300 μL of ultrapure water to a clean 1.5 mL centrifuge tube, vortex for 30 seconds, and filter through a 0.45 μm filter to obtain the test solution; take 2 μL of the supernatant for analysis, and record the chromatogram;

[0079] The quantitative serial concentrations of rifampicin are: 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5μg / mL,

[0080] The quantitative serial concentrations of rifabutin are: 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2μg / mL,

[0081] The quan...

Embodiment 3

[0091] (1) Matrix effect detection

[0092] Take 90 μL of blank plasma, add 10 μL of mixed standard solutions of different concentrations, vortex and mix, add 10 μL of mixed isotope internal standard working solution, then add 300 μL of acetonitrile, vortex for 1 min, and centrifuge at 12000 r / min for 5 min; absorb 300 μL of supernatant in another Add 300 μL of ultrapure water to a clean 1.5 mL centrifuge tube, vortex for 30 seconds, and filter through a 0.45 μm filter to obtain the test solution; prepare 6 parallel samples, and take 2 μL of the supernatant for analysis.

[0093] Use 40% acetonitrile aqueous solution to prepare low and high working solutions of two concentrations (i.e. the concentrations corresponding to the compounds in the quality control sample in the summary of the invention), and prepare 6 copies in parallel, and take 2 μL for analysis. The results are shown in Table 3.

[0094] Table 3: Matrix Effects of Protein Precipitation Experimental Methods

[009...

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Abstract

The invention discloses a method for simultaneously detecting five anti-tuberculosis drugs (including rifampin, rifabutin, Pyrazinamide, ethambutol and isoniazid) in blood plasma via a UPLC-MS / MS method. Blank blood plasma is weighed precisely, and added with standard working solutions mixed with a series of standard substances, standard working solutions of isotope internal standards in one to one correspondence are added, pre-treatment is carried out in a protein precipitation method, the UPLC-MS / MS method is used for analysis, chromatograms of different samples are obtained, and a standardcurve is established by taking the ratio of an object to be measured and the corresponding internal standard peak area as the abscissa and the concentration of the object to be measured as the ordinate; and blood plasma to be measured is weighed precisely, the standard working solutions of isotope internal standards in one to one correspondence are added, pre-treatment is carried out in the protein precipitation method, the UPLC-MS / MS method is used for analysis, chromatograms of different samples are obtained, and the concentration of the blood plasma sample is calculated by using the standard curve. The method is simple and rapid in operation and high in sensitivity, accuracy and precision, the matrix effect is low, and can satisfy requirements for monitoring the drug concentration of five anti-tuberculosis drugs in clinical application.

Description

[0001] (1) Technical field [0002] The invention relates to a method for detecting five anti-tuberculosis drugs (rifampicin, rifabutin, pyrazinamide, ethambutol, and isoniazid) in blood plasma by ultra-high performance liquid chromatography-mass spectrometry, The invention can be used for monitoring the therapeutic drug concentration of these five drugs, and belongs to the technical field of pharmacokinetic analysis. [0003] (2) Background technology [0004] Rifampicin (rifampin RFP) and rifabutin (LM427, rifabutinRFB) both belong to the rifamycin fungicides, and inhibit the RNA polymerase of Mycobacterium tuberculosis so that the bacteria cannot complete transcription process and death; pyrazinamide (pyrazinamind PZA) is a derivative of nicotine amine, has antibacterial or bactericidal effect, and is a half-effect fungicide; The synthesis of sugar leads to the formation of Mycobacterium tuberculosis cell wall and inhibits the growth of bacteria; isoniazid (INH) causes the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 吴灵洁刘景丰刘小龙郑玲叶珍洁储楠楠刘辉
Owner MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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