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Method for detecting concentration of infliximab in serum and application thereof

A technology of fliximon and liximon, which is applied in the field of mass spectrometry detection, can solve problems such as cross-reaction, and achieve the effects of low cost, high detection sensitivity, and short detection time

Inactive Publication Date: 2019-12-03
江苏锐博医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a method for detecting infliximab in serum by high performance liquid chromatography tandem mass spectrometry, which can effectively improve the specificity and speed of detection, and avoid the possibility of traditional immune methods. Cross-reactivity and interference issues that arise

Method used

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  • Method for detecting concentration of infliximab in serum and application thereof
  • Method for detecting concentration of infliximab in serum and application thereof
  • Method for detecting concentration of infliximab in serum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 standard substance preparation

[0039] Take 0.1 mL of infliximab acetonitrile solution with 8 different concentrations (0.1nmol / L, 1nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 40nmol / L, 60nmol / L, 100nmol / L) To a 1.5mL EP tube, add 0.3mL of the above-mentioned isotope peptide internal standard acetonitrile solution (270nmol / L) and 30mg / mL trypsin 0.5μL, which were stored at low temperature, respectively, mix thoroughly with a vortex mixer, and then use a 1mL pipette Transfer 0.3mL internal standard acetonitrile solution containing isotope peptides to another 1.5mL EP tube with a liquid gun, and then lyophilize with centrifugal concentration and freezing system;

[0040] After lyophilization, add 25 μL of water: acetonitrile: formic acid solution with a volume ratio of 9:1:0.05. The mixed components were centrifuged in a cryogenic centrifuge at 17,000 g at 4°C for 5 min, and the supernatant was taken in a 96-well plate for analysis by liquid chromatography tandem ...

Embodiment 2

[0041] Embodiment 2 high performance liquid chromatography separation

[0042] Using ultra-high pressure liquid chromatography and corresponding mobile phase, the supernatant of the sample prepared in Example 1 was chromatographically eluted and separated on a C18 analytical column, and the desired detection infliximon was separated by controlling the elution conditions anti;

[0043] The separation conditions of high performance liquid chromatography are: Waters ACQUITY UPLC C18Column: pore sizeparticle size 1.7μm, 2.1mm×50mm; cat.#186002350IVD; column temperature: 60℃; injection volume: 10μL; flow rate: 0.3mL / min; Mobile phase composition: A phase is 0.1% formic acid aqueous solution, B phase is 0.1% formic acid acetonitrile solution;

[0044] The elution gradient is shown in Table 1 below:

[0045] Table 1

[0046] serial number time (min) Flow rate (mL / min) A％ B％ curve value 1 0.00 0.30 99.00 1.00 - 2 2.00 0.30 99.00 1.00 6 3 4.0...

Embodiment 3

[0047] Embodiment 3 mass spectrometry detects and makes standard curve

[0048] Infliximab and matrix were separated by liquid chromatography and entered into triple quadrupole mass spectrometer for detection, using the reaction monitoring mode in triple quadrupole mass spectrometer to specifically detect the content of infliximab, and draw a standard curve picture;

[0049] According to different chromatographic elution times, different detection windows and parameters are set. The detection parameters are first optimized with standards, and then the multiple reaction monitoring mode in the triple quadrupole mass spectrometer and the optimized specific mass spectrometry parameters are used to specifically detect Inflix. Ciximab and isotopic peptide internal standards.

[0050] The mass spectrometer is Waters Xevo TQD IVD (Waters, Milford, MA); the mass spectrometry detection conditions are as follows: electrospray needle voltage: 2.5kV, desolvation gas flow rate: 800L / h, des...

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Abstract

The invention discloses a method for detecting concentration of infliximab in serum by high-performance liquid chromatography tandem mass spectrometry and application thereof. The method specificallycomprises the following steps of preparation of standard products, enzymatic digestion reaction, high-performance liquid chromatography separation, tandem mass spectrometry detection, and detection ofinfliximab in serum. The invention also discloses application of the method for detecting concentration of infliximab in serum by high-performance liquid chromatography tandem mass spectrometry. Theinvention uses acetonitrile for protein precipitation, and uses chromatographic tandem triple quadrupole mass spectrometer for detection after the extraction, which is simple in pretreatment steps, can effectively remove serum matrix interference, and has good specificity. In addition, the detection is performed by high performance liquid chromatography tandem mass spectrometer, and infliximab inserum is qualitatively and accurately quantified at the same time, thereby having short detection time, high throughput, high detection sensitivity, good specificity and low cost.

Description

technical field [0001] The invention belongs to the technical field of mass spectrometry analysis and detection, and specifically relates to a method for detecting the concentration of infliximab in serum and its application, and more specifically, relates to a detection method for detecting infliximab in serum by using high performance liquid chromatography mass spectrometry series detection method Liximab concentration and application of this detection method. Background technique [0002] Inflammatory diseases are a great threat to human health, and tumor necrosis factor-α (TNF-α) has played a key role in a series of inflammatory diseases, therefore, TNF-α inhibitors have become the treatment of various The mainstay of treatment for inflammatory diseases. Infliximab is the first anti-TNF-α human-mouse chimeric monoclonal antibody approved by the FDA. It has been used clinically since the mid-1990s and has a good effect on many diseases. However, there are still some pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/06G01N30/88G01N2030/045G01N2030/065G01N2030/8822
Inventor 徐雪峰
Owner 江苏锐博医疗科技有限公司
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