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Method for Simultaneously Detecting Five Steroid Hormones in Serum

A steroid hormone and serum technology, applied in the field of analysis and detection, can solve the problems of high reagent cost, matrix interference, and low detection limit, and achieve the effects of strong anti-matrix interference, removal of matrix interference, and reduced cost of consumables

Active Publication Date: 2018-09-21
GUANGZHOU BIOHOP TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] At present, immunoassays are commonly used in China to detect different steroid hormones: such as testosterone, 4-androstenedione, 11-deoxycortisol, cortisone, and hydrocortisone, etc., but immunoassays generally have strong cross-reactivity and specificity. However, the LC-MS / MS method is currently widely accepted as the gold standard method for the quantitative detection of small molecules. Its advantages lie in high sensitivity, low detection limit, good selectivity and accuracy. and high specificity, it is able to distinguish structurally similar compounds
However, due to the wide concentration range of these five hormones in the human body, some concentrations are high, and some concentrations are low, especially the testosterone concentration of women and children, and these steroid hormones mainly exist in the form of protein binding, and there are a large number of high concentrations in the complex composition of human blood. Affinity binding protein, so sampling mass spectrometry detection but still need to solve the following problems: 1) serious matrix interference, poor specificity, low sensitivity; 2) improve detection efficiency, use the same method to detect these five steroid hormones at the same time; 3) different There is a large difference in the concentration of the compound or a large difference in the concentration of the same compound in different populations, requiring a wide linear range
[0017] However, these two methods have the following problems: Method 1 has complex pre-treatment, long processing time (about 2 hours for one sample), low throughput, high reagent cost, and high requirements for operators; Method 2 requires a large investment in equipment and online extraction The cost of consumables is high

Method used

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  • Method for Simultaneously Detecting Five Steroid Hormones in Serum
  • Method for Simultaneously Detecting Five Steroid Hormones in Serum
  • Method for Simultaneously Detecting Five Steroid Hormones in Serum

Examples

Experimental program
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Effect test

Embodiment 1

[0080] The detection method of five steroid hormones of the present embodiment comprises the following steps:

[0081] 1. Sample pretreatment

[0082] Take 200 μL sample (standard curve point and quality control synchronous processing) in a 2.0 centrifuge tube, add 400 μL internal standard working solution ( 13 C3-testosterone 4mg / L, d4-cortisol 4mg / L, D5-11-deoxycortisol 16mg / L) and 13 C3-androstenedione stock solution (8 mg / L acetonitrile solution), high-speed shaking and mixing for 3 minutes; add 800uL tert-butyl methyl ether, high-speed shaking and mixing for 3 minutes; centrifuge at 4°C, 14000r for 10 minutes; take 1 mL of supernatant in 1.5 mL In the centrifuge tube, blow dry with nitrogen at 40°C; reconstitute 100uL of the reconstitution solution and shake at high speed for 3min; centrifuge at 10,000r for 5min at 4°C; transfer 80μL of the centrifuged sample to a sample bottle for detection on the machine.

[0083] 2. Enrichment, separation and detection

[0084] The ...

Embodiment 2

[0109] Embodiment 2 methodological verification experiment

[0110] In this example, methodological verification experiments are carried out for the detection methods of the five steroid hormones in Example 1.

[0111] 1. Precision experiment

[0112] 1.1 Intra-assay: The actual serum samples were spiked to the low, medium and high levels respectively, and the obtained samples were subjected to the intra-assay precision experiment; 20 samples of each concentration level were processed in parallel, and each Sample 1 time; calculate the mean value and relative standard deviation (RSD) of the test results.

[0113] 1.2 Inter-assay precision (Inter-assay): Take the pre-prepared low, medium and high levels of quality control products, and conduct inter-assay precision experiments; samples of each level are made 4 parallels, that is, 4 groups are detected The data were continuously measured for 5 days; the mean value and relative standard deviation (RSD) of the test results were c...

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Abstract

The invention relates to a method for simultaneously detecting five kinds of steroid hormones in serum. The five kinds of steroid hormones are respectively testosterone, androstenedione, 11-deoxycortisol, cortisol and cortisone. The method for simultaneously detecting the five kinds of steroid hormones in the serum comprises the following steps: sample pretreatment: adding an acetonitrile solution of an internal standard substance in a serum sample for carrying out protein precipitation, then adding tert-butyl methyl ether for extracting, blow-drying supernatant, adding a combination solution, and taking the supernatant, thus obtaining a to-be-detected sample; enrichment, separation and detection: carrying out enrichment, separation and detection on the to-be-detected sample by adopting a two-dimensional liquid chromatography-tandem quadrupole mass spectrometer. According to the method for simultaneously detecting the five kinds of steroid hormones in the serum, disclosed by the invention, the method is adopted for simultaneously detecting five kinds of compounds of which the concentration ranges are inconsistent in the serum, and the time for simultaneously detecting the five kinds of the compounds is about 8.5 to 12.0 minutes; the method for simultaneously detecting the five kinds of steroid hormones in the serum is low in cost, high in flux, high in precision degree and strong in specificity.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting five steroid hormones in serum. Background technique [0002] Testosterone is a steroid hormone that is secreted by the testes in men or the ovaries in women, and a small amount by the adrenal glands, but in postmenopausal women, the secretion of testosterone from the ovaries is significantly reduced. The molecular weight of testosterone is 288.42g / moL, and its structural formula is as follows. Most testosterone is bound to sex hormone-binding globulin (SHBG), a small amount is bound to albumin, and a small portion exists in free form. All non-SHBG-bound testosterone is considered bioavailable. During childhood, excess testosterone induces precocious puberty in boys and virilization in girls. In adult women, excess testosterone can cause varying degrees of virilization, including hirsutism, acne, oligomenorrhea, or infertilit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N30/08G01N30/14G01N30/34G01N30/72
CPCG01N30/08G01N30/14G01N30/34G01N30/72G01N30/88G01N2030/8822
Inventor 何翠红李玉冬梁田苗季赖坤元熊晓明
Owner GUANGZHOU BIOHOP TECH INC
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