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Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample

A technology of steroid hormones and samples, applied in the field of analysis and detection, can solve the problems of difficulty in obtaining blood matrix, high cost and complexity, and achieve the effects of saving sampling volume, low price and simple operation.

Active Publication Date: 2021-01-08
GUANGZHOU KINGMED DIAGNOSTICS CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Since steroid hormones are endogenous hormones, they also have a certain distribution in the human body or animals. If human or animal blood is used as a substitute matrix, complex, cumbersome, and time-consuming pretreatments are often required to remove the background before use. At the same time Commercial blood matrix is ​​also difficult to obtain and the cost is high

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  • Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample
  • Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample
  • Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample

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Effect test

Embodiment 1

[0090]This embodiment provides a method for measuring multiple steroids, and the method includes the following steps:

[0091]A. Configure standard curve:

[0092]Blank substrate: Take different proportions of methanol and water in a 35% methanol aqueous solution, mix well and remove bubbles by ultrasonic.

[0093]Using 35% methanol aqueous solution as a blank matrix, 17-OHP was prepared with 271nmol / L, A4 at 41.7nmol / L, cortisol at 600nmol / L, 21-DF at 38.5nmol / L, and 11-DF at 27.1nmol / L. High-concentration samples are diluted one by one to obtain curve points of different concentrations.

[0094]B. Sample pretreatment method

[0095]1) Pipette 100μL of sample into a 2.0mL centrifuge tube, add 50μL of internal standard, add 800μL-1200μL of extraction solution methyl tert-butyl ether (MTBE), vortex vigorously for 5min;

[0096]2) Centrifuge at 10000rpm and room temperature for 5min;

[0097]4) Freeze at -70°C for 60 minutes, transfer the upper layer to a 2.0mL centrifuge tube, add 350μL of 0.1wt% ammonia...

Embodiment 2

[0142]Because the content of different steroid hormones in the serum is very different, some steroid hormones are very low, such as 11-deoxycortisol. In order to improve the detection sensitivity of the analyte, the method of increasing the sampling volume or injection volume can be used. The method generally uses a sampling volume of at least 200μL or more, and an injection volume of 20μL~50μL. Due to the high extraction efficiency and high cleanliness of the sample during the pretreatment process, this method only needs a sampling volume of 100μL and an injection volume of 20μL for 5 types The determination of steroid hormones has a wide linear range and a low limit of quantification. The sampling volume and injection volume can be adjusted according to the needs of clinical applications to further expand the linear range and reduce the limit of quantification to improve detection sensitivity.

Embodiment 3

[0144]In order to achieve accurate determination of low-level hormones (such as 21-deoxycortisol), high-sensitivity instruments are needed. However, when measuring high-level hormones at the same time, there will be a problem of detector saturation, which affects the concentration of high-concentration samples. range. This method adopts the AB SCIEX API 5500 triple quadrupole mass spectrometer. Through the optimization of the collision energy of the mass spectrometer parameters, the response of the hormone items with different human content on the mass spectrometer is similar, and the determination of high and low concentration samples is taken into account. At the same time, due to the collision energy It is the voltage parameter applied to the collision cell to cause the components of the analyte to fragment in the mass spectrum, which has a greater impact on the performance of the project. If the adjustment range is too large, the signal may be unstable during the detection proce...

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Abstract

The invention relates to a pretreatment method, a detection method and a kit for simultaneously detecting multiple steroid hormones in a blood sample. The pretreatment method comprises the following steps: 1), preliminarily extracting the steroid hormone in a serum sample by using methyl tert-butyl ether; 2), performing centrifuging, taking supernatant, and carrying out sufficient alkali washing with an alkaline solution; and 3), performing centrifuging after alkali washing, taking supernate, performing freezing and blow-drying, and redissolving the obtained solid components to obtain a to-be-detected solution for simultaneously detecting various steroid hormones. According to the method, at least five steroid hormones of 17-hydroxy progesterone, androstenedione, cortisol, 21-deoxycortisoland 11-deoxycortisol can be separated at the same time, the separation effect is good, and the operation is simple and convenient.

Description

Technical field[0001]The present invention relates to the technical field of analysis and detection, and in particular to a pretreatment method, detection method and kit for simultaneous detection of multiple steroid hormones in blood samples.Background technique[0002]Congenital adrenal hyperplasia (CAH) is a group of diseases caused by enzyme defects in the pathway of adrenal cortex hormone synthesis, and is an autosomal recessive genetic disease. Enzyme defects include 21-hydroxylase, 11β-hydroxylase, 3-steroid dehydrogenase, 17-hydroxylase deficiency, etc. Among them, 21-hydroxylase deficiency (21-OHD) is the most common type of CAH (90%-95%), most of which are caused by mutations in the CYP21A2 gene encoding 21-hydroxylase. The second is 11β-hydroxylase deficiency (11β-OHD), which accounts for 5% to 8%. Others are very rare.[0003]CAH mainly affects the production of cortisol and aldosterone, and cortisol affects the secretion of sex hormones, causing virilization in children. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88Y02P20/55
Inventor 陈秀如佘旭辉赵蓓蓓韦兰清沈长宇潘燕芳
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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