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
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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
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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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