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Method for detecting steroid hormone in blood

A technology for steroid hormones and detection methods, applied in the field of steroid hormone detection in blood, can solve the problems of lack of standardized operation guidance, difficulty in achieving accurate quantification, and uneven detection results, achieving high accuracy, short time-consuming, heavy-duty and other problems. good effect

Active Publication Date: 2022-01-14
GUANGZHOU KINGMED DIAGNOSTICS CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no commercial kit at present, and medical laboratories generally use self-built projects for CAH secondary screening
However, during the screening process, the preparation of dried blood spots and the pretreatment of blood spot samples were carried out according to conventional methods, and it was found that the test results had problems such as inhomogeneity, instability, and poor accuracy, and there is currently no standardized operation guidance for related screening.
At the same time, there are many steroid hormone isomers in the human body, their chemical properties are close, and the chromatographic peak time is often relatively close. It is difficult to achieve a good separation of various steroid hormone isomers by conventional methods, so it is difficult Realize accurate quantification

Method used

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  • Method for detecting steroid hormone in blood
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  • Method for detecting steroid hormone in blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] (1) Reagent configuration

[0065] Stock solution preparation: prepare 17OHP (17α-hydroxyprogesterone), A4 (androstenedione), F (cortisol), 21-DF (21-deoxycortisol), 11-DF with a concentration of 1mg / mL in methanol (11-deoxycortisol) single standard stock solution;

[0066] Red blood cell washing: use heparin anticoagulated whole blood, centrifuge at 3000rpm for 5min, discard the upper layer of plasma, add an equal volume of normal saline solution, use a blood mixer to mix for 5min, centrifuge at 3000rpm for 5min, absorb the supernatant, and discard , repeat this operation 5 times to obtain red blood cells;

[0067] Serum standard curve configuration: use 30% methanol water to dilute the stock solution, use hormone-free serum as the blank matrix, and use a volumetric flask to configure a series of standard working curves. The concentration range of the curve is: 17OHP: 6-2225nmol / L, A4 :2-680nmol / L, F: 6-1400nmol / L, 11-DF: 4-720nmol / L, 21-DF: 6-1200nmol / L;

[0068] D...

Embodiment 2

[0106] Four kinds of organic solvents of methanol, acetonitrile, tert-butyl methyl ether, and n-hexane were respectively used as extraction agents, and according to the sample pretreatment method of step (2) of Example 1 of the present invention, samples with three concentration levels of low, medium, and high were processed. Three samples were processed in parallel. The result is as Figure 1-5 shown, from Figure 1-5 It can be seen that when methanol / acetonitrile (1:0.3) is used as the extraction solvent in Example 1, the peak areas of the five steroid hormones are the highest, the instrument response is the strongest, and the extraction efficiency is the highest, followed by methanol, while acetonitrile, tert-butyl methyl ether, n-hexane The extraction efficiency is poor when alkane is used as the extraction solvent.

Embodiment 3

[0108]Get the samples of low, medium and high concentrations, according to the sample pretreatment method of step (2) of embodiment 1 of the present invention, each sample is processed in parallel 3, parallel processing 4 groups, the extraction time of the first group is 30min (i.e. Example 1), the second group of extraction time is 60min, the third group of extraction time is 90min, the fourth group of extraction time is 120min, adopt Passing&Bablokregression to analyze, the results are as shown in Table 8, Cusum test confirms that the linear regression bias has no statistics Meaning, the correlation coefficient of Spearman's is all>0.80, shows that the test results obtained by different extraction times are highly correlated, and the test results obtained by the 30min extraction time and the 60min, 90min, and 120min extraction times have no significant difference. In the present invention, by Vibration heating extraction, the best extraction time is 30min, a shorter time can ...

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Abstract

The invention relates to a method for detecting steroid hormones in blood. The method comprises the following steps: preparation of a standard curve dried blood spot sample: preparing red blood cells by using whole blood; by taking hormone-removed serum as a blank matrix, adding standard substances such as 17alpha-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycortisol and 11-deoxycortisol solutions to prepare a series of serum standard curve working solutions; mixing the red blood cells with the serum standard curve working solution, and preparing a standard curve dried blood spot sample by using the obtained mixed solution; pretreatment of the dried blood spot sample: taking the dried blood spot sample, adding an internal standard-containing extraction liquid, extracting, centrifuging, drying, adding a reconstitution fluid, centrifuging, and determining by adopting liquid chromatography-tandem mass spectrometry. The detection method disclosed by the invention is simple in pretreatment operation, short in time consumption, high in stability and good in reproducibility, and various steroid isomers can be efficiently separated at the same time.

Description

technical field [0001] The invention relates to the field of analysis, in particular to a method for detecting steroid hormones in blood. Background technique [0002] Congenital adrenal hyperplasia (CAH) is a more common cause of adrenal insufficiency, which is a group of autosomal recessive inheritance whose main feature is cortisol synthesis disorder due to the deficiency of a certain enzyme in steroid hormone anabolic metabolism Metabolic diseases, the overall incidence of live births is about 1 / 20000 to 1 / 10000. CAH is divided into 6 types according to enzyme defects, the most common of which is 21-hydroxylase deficiency, accounting for about 95%; followed by 11-β hydroxylase deficiency, accounting for about 3% to 5%, 17α-hydroxylase deficiency Enzyme / 17,20-lyase deficiency and 3β-hydroxydehydrogenase deficiency account for about 1% respectively, and other types are rare. [0003] CAH mainly affects the production of cortisol and aldosterone, and cortisol affects the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/72G01N30/8634G01N2030/045
Inventor 赵蓓蓓佘旭辉陈秀如董衡李卓阳蔡舒婷韦兰清
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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