Detecting method and detecting kit for immunosuppressive agents in dried blood spots

An immunosuppressant and detection method technology, applied in the field of clinical testing, can solve the problem of no immunosuppressant detection method in dried blood slices, and achieve the effects of reducing the risk of pathogen infection, good stability at room temperature, easy storage and transportation

Inactive Publication Date: 2018-12-18
易达精准(杭州)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Dried blood filter paper sample collection technology has incomparable advantages over whole blood samples, including better stability at room temperature, minimal invasiveness, lower cost, easy storage and transportation, and reduced potential risk of pathogen infection. Methods for the detection of immunosuppressants

Method used

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  • Detecting method and detecting kit for immunosuppressive agents in dried blood spots
  • Detecting method and detecting kit for immunosuppressive agents in dried blood spots
  • Detecting method and detecting kit for immunosuppressive agents in dried blood spots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The simultaneous detection method of four kinds of immunosuppressants in dried blood film comprises the following steps:

[0069] (1) Preparation of dried blood slice standard curve sample: Take 495uL healthy volunteer whole blood and 5uL mixed standard solution (200ug / ml cyclosporine A, 1ug / ml tacrolimus, 1ug / ml rapamycin, 1ug / ml everolimus), and then diluted with whole blood of healthy volunteers to make the concentration of cyclosporine A 10, 20, 100, 200, 500, 1000, 1500, 2000ng / ml; tacrolimus , rapamycin, and everolimus concentrations were 0.5, 1, 5, 10, 20, 50, 75, and 100 ng / ml. Take 40 μl of the prepared samples above and blot them on whatman903 filter paper to dry.

[0070] (2) Preparation of samples to be tested: take fresh whole blood and stain it on whatman903 filter paper to dry.

[0071] (3) Analysis process: (a) punch a blood spot sample with a 6mm hole puncher to contain an internal standard solution (the internal standard solution contains cyclosporine...

Embodiment 2～5

[0086] Compared with Example 1, the extract was replaced by pure water, 20% methanol, 50% methanol, and pure methanol from 80% methanol aqueous solution.

[0087] Quality control samples of the same concentration were taken, and the immunosuppressant in the dried blood slices was extracted according to the process described in Example 1. After ultrasonic extraction for 30 minutes, the extract was obtained by centrifugation at 15,000 g for 5 minutes. After being dried and reconstituted, it can be used on the machine, and a good signal response value can be obtained. Among them, the signal response value in Example 1 (the extract solution is 80% methanol aqueous solution) is the strongest.

Embodiment 6～10

[0089] The eluent in Example 1 was replaced with the components shown in Table 3, and the others were the same as in Example 1.

[0090] Table 3: Eluent formulations

[0091] Example

[0092] Take quality control samples of the same concentration, respectively use the eluent of the formula shown in Table 3, and detect the content of the immunosuppressant according to the liquid phase tandem mass spectrometry method described in Example 1. Examples 6-10 can obtain relatively ideal detection The recovery rate of each analyte can meet the detection requirements stipulated by the US Food and Drug Administration.

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Abstract

The invention discloses a detecting method for immunosuppressive agents in dried blood spots, and further discloses a detecting kit for the immunosuppressive agents in the dried blood spots. The detecting method comprises the steps that the standard curve dried blood spots and the to-be-detected dried blood spots are mixed with an extracting solution and an internal standard solution correspondingly and then extracted, centrifuging is conducted, and supernate is taken; the supernate is blown dry and then redissolved through a redissolving solution, and detecting is conducted through a tandem mass spectrum method; according to the peak exceeding area specific value of standard samples to an internal standard object and the concentration of the standard samples, linear regression is conducted to obtain a standard curve equation of the immunosuppressive agents; the peak exceeding area specific value of to-be-detected samples to the internal standard object is substituted into the standardcurve equation, and the content of the immunosuppressive agents in the to-be-detected samples is calculated; the extracting solution is a water solution of methyl alcohol and/or acetonitrile; and theimmunosuppressive agents are one or more of cyclosporine A, tacrolimus, sirolimus and everolimus. The detecting method can effectively detect the four types of immunosuppressive agents simultaneously, and thus the detecting efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of clinical detection, in particular to a method for detecting immunosuppressants in dried blood slices and a detection kit. Background technique [0002] Immunosuppressants are drugs that have an inhibitory effect on the body's immune response. They can inhibit the proliferation and function of cells related to the immune response (macrophages such as T cells and B cells), and can reduce the antibody immune response. Immunosuppressants are mainly used for organ transplantation anti-rejection and autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, skin mycosis, membranous glomerulonephritis, inflammatory bowel disease and autoimmune hemolytic anemia. [0003] Currently, the control of the immunosuppressive state is a thorny issue in drug therapy after organ transplantation. Commonly used immunosuppressants mainly include: glucocorticoids (hydrocortisone, prednisone, prednisolone, methylpr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88
Inventor 李士敏袁京群吴筱丹葛志伟朱亚尔李家睿
Owner 易达精准(杭州)科技有限公司
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