Method for detecting four progestational hormones in serum

Abstract

The invention provides a method for detecting four progestational hormones in serum. The four progestational hormones are 17alpha-hydroxypregnenolone, 17alpha-hydroxyl progesterone, progesterone and pregnenolone; and the method comprises the following steps: adding a methanol solution containing an internal standard substance in a serum sample, performing protein precipitation, centrifuging and taking supernatant, adding derivatization reagent to perform derivatization reaction, cooling to room temperature and then adding tert-butyl methyl ether, centrifuging and taking the supernatant, thereby obtaining a to-be-detected sample, wherein the internal standard substance is 17alpha-hydroxypregnenolone internal standard substance, the 17alpha-hydroxyl progesterone internal standard substance, the progesterone internal standard substance and pregnenolone internal standard substance; enriching, separating and detecting: performing enriching, separation and detection on the to-be-detected sample by adopting a two-dimensional liquid chromatography tandem quadrupole mass spectrometer. Through the method provided by the invention, the matrix interference can be effectively removed, and the method has the features of being strong in specificity and matrix interference resistance, low in quantitation and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, in particular to a method for detecting four progestins in serum. Background technique [0002] Cholesterol generates pregnenolone under the action of C20 and C22 side chain hydroxylase, and pregnenolone first passes through 3β-hydroxyl steroid dehydrogenase and Δisomerase to remove the double bond between C5 and C4 to generate progesterone , the progesterone continues to pass through the Δ pathway to continue to generate 17α-hydroxyprogesterone; pregnenolone can also be converted into 17α-hydroxypregnenolone, pregnenolone, and progesterone through the action of 17α-hydroxylase (CYP17A1). Ketone, 17α-hydroxyprogesterone has an important physiological role. [0003] Progesterone, a natural progesterone secreted by the corpus luteum of the ovary, has a significant morphological effect on the endometrium stimulated by estrogen in vivo, and is necessary for maintaining pregnancy. The...

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Problems solved by technology

[0008] At present, there are few detection methods for 17α-hydroxypregnenolone and pregnenolone in China. The detection methods of progesterone and 17α-hydroxyprogesterone are generally detected by immunoassay, but the immunoassay generally has strong cross-reactivity and poor specificity. , low sensitivity, low accuracy and other issues, and 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 can distinguish structurally similar compounds

However, due to the wide concentration range of these four progestins in the human body, some of which are high and some are low, and these steroid hormones mainly exist in the form of protein binding, and there are a large number of high-affinity binding proteins in the complex composition of human blood, so mass spectrometry However, the following problems still need to be solved: 1) serious matrix interference, poor specificity, and low sensitivity; 2) improve the detection efficiency, and use the same method to detect these four progestogens at the same time; 3) the concentration of different compounds varies greatly or the same compound The concentration of different groups of people varies greatly, requiring a wide linear range

[0012] 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

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