Method for detecting steroid hormones in biological sample and kit used in method
A technology for steroid hormones and biological samples, which is applied to the detection method of steroid hormones in biological samples and the field of kits used in the method, can solve the problems of reduced MS detection sensitivity, low ionization efficiency of hormone molecules, etc. Ionization efficiency, simple and straightforward preparation
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Embodiment 1
[0083] Feasibility Analysis of Determination of Steroid Hormones in Plasma by HA-D / MALDI-MS. Proceed as follows:
[0084] 1) Standard steroid hormones were prepared with methanol to 10 μg mL -1 (E1, T, and Prog are 37.02, 34.70, and 31.82 μM, respectively), take 500 μL, and take an equal volume of methanol solution at the same time, and dry it in a freeze dryer.
[0085] 2) Add 500 μL of 300 mM HA hydrochloride dissolved in 50% methanol to the hormone standard and methanol respectively. At the same time, take the same drained hormone standard and add 500 μL of 50% methanol. After 1 h at 60°C, the derivative product was sucked dry using a freeze dryer. The dried product containing the steroid oxime derivative was redissolved in 20 μL of 50% methanol. derivatization of hormones, and subsequent dehydroxylation steps such as figure 1 shown.
[0086] 3) Dissolve 1.5 μL of the solution with 1.5 μL of DHB [acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v), 10 mg mL -1...
Embodiment 2
[0090] Optimization of the workflow for the analysis of steroid hormones in plasma. Proceed as follows:
[0091] 1) Take plasma samples and mix equal volumes to form a mixed plasma sample. Take 400 μL of each plasma sample, add 800 μL of methanol to precipitate protein, centrifuge at 3500 rpm at 4 °C for 15 min, then transfer 1 mL of supernatant to a new tube, and dry in a freeze dryer.
[0092] 2) Add 500 μL of HA hydrochloride dissolved in 50% methanol, and the derivatization temperature is 60°C. In order to obtain excellent analytical performance, we optimized the key points of this method, including the concentration of HA hydrochloride, derivatization time and the type of SPE chromatographic column. The HA concentrations were 1, 10, 50, 100, 600 mM respectively; the derivatization times were 1, 2, 3, 4, 5 hours, respectively. After treatment with a derivatizing reagent, the reaction product was subjected to SPE. The SPE columns are H, P, and P-H, representing SPE colum...
Embodiment 3
[0097] Comparison of the derivatization efficiencies of the carbonyl steroid hormones HA and GT. It is characterized in that 6.94mM, 69.39nM and 17.35nM T are derivatized with HA and GT respectively, the HA-D conditions are as mentioned above, 300mM HA, incubated for 1 hour, and purified by HLB Oasis SPE column. For GT derivatization, prepare 1 mL of 60 mM GT dissolved in 50% methanol, 5% acetic acid, and incubate at 85 °C for 4 h. To compare the performance of GT derivatization and HA derivatization, other experimental procedures were the same.
[0098]We analyzed the same concentrations of steroid hormone standards for HA derivatization and GT derivatization, respectively, and compared the derivatization performance of the two by MALDI-MS. Figure 5 The mass spectrum in shows that after HA derivatization ( Figure 5 a) and GT derivatization ( Figure 5 b) After that, a strong signal was observed at 6.94mM T, T-oxime m / z was 288.2, T-hydrazone m / z was 402.3, but, as Figu...
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