Methotrexate detection method

A technology for methotrexate and detection methods, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of long sample detection time, time-consuming, complicated pre-treatment, etc.

Pending Publication Date: 2021-02-26
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection of high performance liquid chromatography usually requires more complicated pretreatment of the sample to be tested, which consumes more time, resulting in a longer sample detection time.

Method used

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  • Methotrexate detection method
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  • Methotrexate detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Embodiment 1: prepare the standard solution of series concentration

[0140](a) Preparation of standard stock solution:

[0141] Accurately weigh 4.45mg of methotrexate standard substance and place it in a 2mL volumetric flask, dissolve it with 0.1M aqueous sodium hydroxide solution, and set the volume to 2mL to obtain a standard stock solution, which is stored at -80°C and has a validity period of 24 months.

[0142] (b) Preparation of standard working solution

[0143] Take an appropriate amount of the standard stock solution in step (a), dilute and mix it with an aqueous solution containing 50% methanol as a diluent to obtain a standard working solution containing 100-50000ng / mL methotrexate, and store it at -80°C. Valid for 6 months;

[0144] Among them, the standard working solution with different concentrations is a series of solutions containing methotrexate: 100ng / mL, 400ng / mL, 1000ng / mL, 2000ng / mL, 5000ng / mL, 10000ng / mL, 20000ng / mL, 50000ng / mL .

[0145] (...

Embodiment 2

[0151] Embodiment 2: fitting standard curve equation

[0152] The eight kinds of standard solutions in Example 1 were detected respectively by liquid chromatography-mass spectrometry, and the chromatograms of eight kinds of standard solutions of methotrexate with different concentrations were obtained.

[0153] From the standard solution chromatogram of above-mentioned methotrexate, obtain respectively the corresponding peak areas of methotrexate and internal standard in eight kinds of standard solutions, with the methotrexate obtained in the chromatogram of the standard solution of above-mentioned each concentration The ratio of the peak area of ​​the methotrexate and the chromatographic peak area of ​​the internal standard is used as the ordinate y1 of the standard curve equation, and the concentration in the above-mentioned methotrexate standard working solution and the concentration of the internal standard are used as the abscissa x1 of the standard curve equation, Perfor...

Embodiment 3

[0166] Embodiment 3: the pretreatment of sample to be tested

[0167] 3.1 Take at least 2mL of blood to be processed, centrifuge at a centrifugation speed of 3500rpm for 10min, take the supernatant serum or plasma as the first supernatant, and store the above serum or plasma at -20°C until analysis.

[0168] 3.2 Use a pipette gun to pipette 10 μL of the internal standard working solution in Example 1 into a 1.5 mL centrifuge tube, then add 100 μL of the serum or plasma in step 3.1, vortex and mix at 2000 rpm for 0.5 min, and then add the precipitate Protein reagent 300 μL aqueous solution containing 6% sulfosalicylic acid, after vortex mixing at 2000 rpm for 3 minutes, and then high-speed centrifugation at 12000 rpm for 10 minutes, the second supernatant obtained is the sample to be tested.

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Abstract

The invention provides a methotrexate detection method, and the method comprises the following steps: preparing standard solutions with at least three concentrations of methotrexate and internal standard substances, wherein the quantities of the internal standard substances in the standard solutions are the same; detecting each standard solution by using a liquid chromatograph-mass spectrometer under a detection condition to obtain a first detection result corresponding to the standard solution; fitting a standard curve equation of methotrexate according to each first detection result, the concentration of methotrexate in the standard solution and the concentration of the internal standard substance; taking a first supernatant of the sample to be treated after centrifugation; adding an internal standard substance and a precipitated protein reagent into the first supernatant, carrying out vortex mixing uniformly, centrifuging, and taking a centrifuged second supernatant as a sample to be detected; detecting the to-be-detected sample by utilizing the liquid chromatograph-mass spectrometer under the detection condition to obtain a second detection result of the to-be-detected sample;based on the standard curve equation and the second detection result, obtaining the concentration of methotrexate in the to-be-detected sample. The scheme can shorten the sample detection time.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection method of methotrexate. Background technique [0002] Methotrexate is a folate reductase inhibitor, orange-yellow crystalline powder, easily soluble in dilute alkali, acid or alkali metal carbonate solution, slightly soluble in dilute hydrochloric acid, almost insoluble in water, ethanol, chloroform , Ether. [0003] At present, the method usually used to detect the content of methotrexate in a sample is high performance liquid chromatography. However, HPLC detection usually requires more complex pretreatment of the sample to be tested, which consumes more time, resulting in a longer sample detection time. Contents of the invention [0004] The invention provides a method for detecting methotrexate, which can shorten the sample detection time. [0005] In order to solve the above problems, the embodiment of the present invention provides a method for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88G01N30/89
CPCG01N30/06G01N30/88G01N30/89
Inventor 贾永娟刘杏立刘春冉倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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