CN-122017085-A - Method for simultaneously detecting multiple steroid hormones in serum

Abstract

A method for simultaneously detecting various steroid hormones in serum comprises the steps of extracting the hormones in the serum through a liquid-liquid extraction method, adding a derivatization reagent into the extracted hormones to carry out oximation reaction, wherein the derivatization reagent comprises quaternary ammonium oxamine, injecting the hormone subjected to the oximation reaction into a two-dimensional liquid chromatography-tandem mass spectrometry system to carry out detection, enriching the hormone on a one-dimensional chromatographic column, and then carrying out back flushing transfer of the enriched hormone onto the two-dimensional chromatographic column for analysis. The method combines the back flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry with the derivatization reaction, can detect various steroid hormones including progestogen and androgen at the same time, and has the advantage of high detection sensitivity.

Inventors

• ZHAO LIBO
• ZHANG XIANHUA
• LI JUEYU
• LIU LINGLING
• XIONG XIN
• XU LEI
• JIA YUQI
• YANG LI
• ZHAO RONGSHENG

Assignees

• 北京大学第三医院（北京大学第三临床医学院）

Dates

Publication Date

20260512

Application Date

20260330

Claims (10)

1. 1. A method for simultaneously detecting a plurality of steroid hormones in serum, wherein the method comprises: extracting hormone in serum by liquid-liquid extraction; adding a derivatizing reagent to the extracted hormone to perform an oximation reaction, wherein the derivatizing reagent comprises quaternary ammonium oxamine; injecting the hormone after the oximation reaction into a two-dimensional liquid chromatography-tandem mass spectrometry system for detection, wherein the hormone is enriched on a one-dimensional chromatographic column, and then, carrying out backflushing transfer on the enriched hormone to the two-dimensional chromatographic column for analysis.
2. 2. The method of claim 1, wherein the packing of the two-dimensional chromatography column comprises adamantyl and/or phenylhexyl groups.
3. 3. The method of claim 2, wherein the volume ratio of adamantyl and phenylhexyl in the two-dimensional chromatography column is 4:1.
4. 4. The method according to claim 1, wherein the detection is performed using a mixed mobile phase consisting of a mobile phase a which is an aqueous ammonium formate solution containing 0.1% (v/v) formic acid at a concentration of 10mM and a mobile phase B which is acetonitrile containing 0.1% (v/v) formic acid; In the gradient elution process, the volume ratio of the mobile phase B in the mixed mobile phase is gradually increased from an initial proportion, and then the mobile phase B is restored to the initial proportion; in the gradient elution process, the elution temperature is 45 ℃, the flow rate is 0.6 mL/min, and the elution time is 21min.
5. 5. The method of claim 4, wherein the hormone is further delivered to the one-dimensional chromatographic column by an auxiliary pump as it is enriched on the one-dimensional chromatographic column.
6. 6. The method of claim 1, wherein the total time of the backflushing transfer is 0.1-5min.
7. 7. The method of claim 1, wherein the derivatizing agent further comprises methanol, water, and formic acid, wherein the concentration of formic acid is 2-15% (V/V).
8. 8. The method according to claim 1, wherein the oximation reaction is carried out at a reaction temperature of 30-50 ℃ for a reaction time of 60-90min.
9. 9. The method according to claim 3, wherein the one-dimensional chromatographic column is a C18 chromatographic column with a specification of 100X 4.6mm and 5 μm, and the two-dimensional chromatographic column with a specification of 100X 3mm and 2.6 μm.
10. 10. The method of claim 1, wherein the steroid hormone is selected from testosterone, dehydroepiandrosterone, dihydrotestosterone, androstenedione, 11 beta-hydroxy testosterone, 11-ketotestosterone, 11-ketoandrostenedione, progesterone, 17-hydroxy progesterone, pregnenolone, 17-hydroxy pregnenolone.

Description

Method for simultaneously detecting multiple steroid hormones in serum Technical Field The application relates to a detection method combining liquid chromatography and tandem mass spectrometry, in particular to a method for simultaneously detecting various steroid hormones in serum. Background Polycystic ovary syndrome is a common disease of the reproductive endocrine system in women of childbearing age, and about 8% -13% of women of childbearing age worldwide suffer from the disease and have become one of the main causes of female infertility. Hyperandrogenic symptoms are a marked characteristic of polycystic ovary syndrome, and a series of endocrine and metabolic disorders such as insulin resistance, ovulation dysfunction and the like are often accompanied by the rise of androgen levels. At present, the source and the action mechanism of the excessive secretion of androgens of polycystic ovary syndrome patients are not completely elucidated, and the sex hormone spectrums of different patients have obvious individual differences, so that clinical treatment schemes are different. Therefore, the method has important clinical significance in accurately and quantitatively detecting the sex hormone of the polycystic ovary syndrome patient. Sex hormones are an important class of steroid hormones in the human body, mainly including progestins, androgens and estrogens. The adrenal glands play a key role in sex hormone synthesis during fetal and pre-pubertal phases, and after pubertal entry, the major synthesis site of sex hormone is converted into the gonad. In clinical practice, the major sex hormones tested for polycystic ovary syndrome patients include testosterone (T), androstenedione (a 4), progesterone (P), and estradiol (E 2), with testosterone and androstenedione levels often appearing to be significantly elevated. However, the sex hormones in human bodies are various, and extremely complex interconversion relations exist among various sex hormones, so that the sex hormone spectrum characteristics of patients with polycystic ovary syndrome are difficult to accurately and comprehensively reflect only by detecting the few hormones. In the last decade, more and more studies have found that 11-oxoandrogens play a key role in a variety of diseases such as polycystic ovary syndrome. In the human body, both 11-oxidized androgens and classical androgens use cholesterol and progestogen as upstream synthesis precursors, the progestogen is firstly converted into classical androgens, and the classical androgens further generate 11-oxidized androgens under the action of CYP11B enzyme. Such 11-oxidases are expected to become important biomarkers for diagnosis of polycystic ovary syndrome, however, the current 11-oxidases detection research on polycystic ovary syndrome patients is still relatively deficient. In clinical practice, immunoassays are still the standard methods for sex hormone detection. Although the method has the advantages of high automation degree and low cost, the inherent limitations affect the accurate quantification of various hormones. Firstly, steroid hormones with similar structures can cross react with detection antibodies, so that positive deviation appears on detection results, analysis precision is reduced, and secondly, the content of various sex hormones (such as dihydrotestosterone and 11-hydroxytestosterone) circulating in human bodies is extremely low, and the concentration of the sex hormones is often lower than the lower limit of the reliable quantitative sensitivity of an immunoassay method. In order to overcome the limitations of the immunoassay in terms of specificity, sensitivity and multi-index simultaneous detection, the research of taking sex hormone detection as a main endpoint should be adopted by a detection method based on mass spectrometry technology, which is clearly proposed in 2013 of journal of clinical endocrine and metabolism. The liquid chromatography-tandem mass spectrometry is used for quantifying the compound according to the molecular mass and characteristic fragment ions, and has the advantages of strong specificity, high sensitivity, capability of simultaneously analyzing various components and no cross reaction interference. In order to further improve the separation efficiency and the detection sensitivity, the two-dimensional liquid chromatography-tandem mass spectrometry technology has the basic principle that two chromatographic columns are connected through a switching valve, fractions eluted by one-dimensional chromatographic columns are circularly collected for a plurality of times and then injected into the two-dimensional chromatographic columns for further separation, and the technology shows excellent performance in the aspects of separation capacity and information acquisition. At present, the two-dimensional liquid chromatography-tandem mass spectrometry technology is widely applied to the fields of natural medicine analysis, proteomics, chiral com