Method and kit for simultaneously detecting five steroid hormones and application thereof

A technology of steroid hormones and isotopes, applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effect of high practical application value

Active Publication Date: 2022-06-10
武汉迈特维尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, LC-MS / MS methods capable of simultaneously detecting five steroid hormones including testosterone, androstenedione, 17α-hydroxyprogesterone, dihydrotestosterone, and dehydroepiandrosterone sulfate have been rarely reported, and the pretreatment method is protein precipitation. Combined with solid-phase extraction, however, the operation of solid-phase extraction is more complicated, the repeatability between batches is difficult to guarantee, and the price is expensive

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  • Method and kit for simultaneously detecting five steroid hormones and application thereof
  • Method and kit for simultaneously detecting five steroid hormones and application thereof
  • Method and kit for simultaneously detecting five steroid hormones and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Sample preparation

[0044] 1. Preparation of standard curve and quality control samples

[0045] Prepare testosterone, androstenedione, 17α-hydroxyprogesterone, dihydrotestosterone, and dehydroepiandrosterone sulfate standard products into a mixed solution, which is used as a standard working solution and a quality control working solution, respectively mixed with 3% BSA in phosphate buffered saline Liquid and negative serum were mixed at a volume ratio of 1:49 to prepare calibrator and quality control samples.

[0046] The 5 steroid hormones have 6 serial concentrations (C1-C6) in the standard product, as shown in Table 1:

[0047] Table 1. Concentrations of 6 series of 5 steroid hormones in standard products (C1~C6)

[0048]

[0049] The 5 steroid hormones have two series concentrations of low (L) and high (H) in quality control products, as shown in Table 2:

[0050] Two serial concentrations of table 2, 5 steroid hormones in quality control products

...

Embodiment 2

[0091] Adopting the internal standard solution system provided in this example not only simplifies the pretreatment operation, but also has a high extraction recovery rate of the five steroid hormones, combined with the high sensitivity and accuracy of liquid chromatography-tandem mass spectrometry, it makes the blood sample species The detection ability of 5 steroid hormones can meet the actual needs of clinical reference intervals at the same time. The internal standard solution system provided in this example contains 25~35mmol / L zinc sulfate (preferably 30mmol / L) for pretreatment, and through the follow-up high performance liquid chromatography tandem mass spectrometry detection verification, testosterone, androstenedione, 17α -Hydroxyprogesterone, dihydrotestosterone, and dehydroepiandrosterone sulfate have the highest sensitivity of 0.015ng / mL, 0.025ng / mL, 0.25ng / mL, 0.15ng / mL, and 2.5ng / mL, respectively.

[0092] In yet another specific embodiment of the present inventi...

Embodiment 3

[0100] 3) Preparation of internal standard: prepare mixed internal standard working solution with methanol, 2 h 3 -T, A4- 13 C 3 , 2 h 8 -17-OHP, 2 h 4 -DHT, 2 h 6 -The concentrations of DHEAS were 100ng / mL, 200ng / mL, 400ng / mL, 2000ng / mL, 10000ng / mL, respectively. Take 240 μL of the prepared internal standard and put it in a 1.5mL sample bottle, put it in a concentrator, concentrate and dry it into a dry powder, and store it at 2-8°C for later use. When in use, take it out from the refrigerator and let it stand at room temperature for 15-20 minutes to equilibrate, then add 240 μL of 50% methanol, mix well and set aside.

[0101] 4) Preparation of internal standard solution: Take 125 mg of zinc sulfate and add 25 mL of 50% methanol to prepare a mixed solution A containing 30 mM zinc sulfate and 50% methanol. Then take 200 μL of the internal standard working solution and add it to 20 mL of the mixed solution A to prepare an internal standard solution with a ratio of 1:...

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Abstract

The invention provides a method and a kit for simultaneously detecting five steroid hormones and application of the method and the kit, and belongs to the technical field of steroid hormone detection. The method comprises the step of detecting a pretreated sample to be detected on the basis of LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). The pretreatment method is optimized, so that accurate detection of five steroid hormones including testosterone, androstenedione, 17alpha-hydroxyprogesterone, dihydrotestosterone and dehydroepiandrosterone sulfate is realized, and the method can be applied to detection items of important steroid hormones in clinical androstenemia. The real level of the real concentration of the five steroid hormones in the body of a subject can be given easily, clinical use or laboratory use is facilitated, and the method has important guiding significance on auxiliary diagnosis of hyperandrogenemia and polycystic ovarian syndrome and has high practical application value.

Description

technical field [0001] The invention belongs to the technical field of steroid hormone detection, and in particular relates to a method for simultaneously detecting five steroid hormones, a kit and an application thereof. Background technique [0002] Polycystic ovary syndrome (PCOS) is a common reproductive endocrine and metabolic disease. . Due to its highly heterogeneous clinical manifestations, it not only seriously affects the reproductive function of patients, but also increases the incidence of estrogen-dependent tumors such as endometrial cancer, and related metabolic disorders include hyperandrogenemia, insulin resistance, and abnormal glucose metabolism , abnormal lipid metabolism, and the incidence of cardiovascular disease will also increase. At this stage, the diagnosis of PCOS is mainly based on the criteria recommended by the expert meeting of the European Society of Reproduction and Embryology and the American Society for Reproductive Medicine in 2003: ① ol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/04G01N30/14G01N30/30G01N30/32G01N30/34G01N30/72G01N30/06
CPCG01N30/02G01N30/04G01N30/14G01N30/30G01N30/32G01N30/34G01N30/7233G01N30/7266G01N2030/027G01N2030/045G01N2030/065G01N2030/3007G01N2030/324G01N2030/342
Inventor 殷文敏陈丽珍姜宽唐堂
Owner 武汉迈特维尔生物科技有限公司
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