CN111398447B

CN111398447B - Kit for detecting 12 steroid hormone in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology

Info

Publication number: CN111398447B
Application number: CN202010172191.5A
Authority: CN
Inventors: 成晓亮; 李美娟
Original assignee: Fujian Pinsheng Wanfu Medical Testing Co ltd; Fuzhou Pinsheng Medical Technology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd; Nanjing Pinsheng Medical Technology Co ltd
Current assignee: Fujian Pinsheng Wanfu Medical Testing Co ltd; Fuzhou Pinsheng Medical Technology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd; Nanjing Pinsheng Medical Technology Co ltd
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2023-05-05
Anticipated expiration: 2040-03-12
Also published as: CN111398447A

Abstract

The invention relates to a kit for detecting 12 steroid hormones in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, which is characterized in that a serum sample is mixed with mixed internal standard solutions of all objects to be detected, the sample does not need derivatization treatment, and the solution to be detected can be obtained by one-step liquid-liquid extraction; by adding electrolyte ammonium fluoride in the mobile phase, the ionization efficiency of certain target compounds can be effectively improved; and then 12 hormones are detected simultaneously by adopting a mass spectrum positive-negative switching scanning mode, so that the detection kit has the advantages of multiple detection types, high sensitivity, strong specificity, low cost, simple and quick pretreatment process, no need of solid phase extraction or derivatization, and capability of completing separation and detection of the 12 hormones within 5.0min, and is provided for clinical health assessment of endocrine diseases.

Description

Kit for detecting 12 steroid hormone in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting 12 steroid hormones in serum by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology.

Background

Steroid hormones are a class of fat-soluble small molecule hormones, also known as steroid hormones. They are produced by cholesterol through a series of enzyme catalytic metabolism, and play an important role in regulating metabolism, immunoregulation, sexual function regulation, growth and development and the like of organisms. Steroid hormones can be classified into progestins, mineralocorticoids, glucocorticoids, estrogens and androgens according to their physiological functions. Progesterone, corticosterone, aldosterone belong to the mineralocorticoids, 17 alpha-hydroxyprogesterone and cortisol belong to the glucocorticoids, estrone and estradiol belong to the estrogens, dehydroepiandrosterone sulfate, dehydroepiandrosterone, testosterone, dihydrotestosterone and androstenedione all belong to the androgens.

Progesterone (P4), also known as progesterone, is derived from the adrenal gland, corpus luteum and placenta. Progesterone detection can determine ovulation period, evaluate infertility, evaluate abnormal uterine bleeding, high risk gestation placenta health status, etc. Pathological increases in progesterone can be seen in toxemia of pregnancy, preeclampsia, essential hypertension, congenital 17-hydroxyprogesterone production disorder, etc.; the pathological decrease of progesterone is found in threatened abortion, ectopic pregnancy, premature labor, amenorrhea, fetal hypoevolutism, stillbirth, infertility, luteal phase insufficiency, and serious adrenal gland or thyroid gland dysfunction. 17 alpha-hydroxyprogesterone and progesterone are the prerequisite for other steroid hormones, and their increased levels contribute to conception.

Aldosterone (ALD) is a mineralocorticoid that acts primarily on the kidneys to reabsorption of sodium ions and water. As a whole, aldosterone is a hormone that enhances the kidney's reabsorption of ions and moisture, and is part of the renin-angiotensin system. The increased aldosterone is found in primary aldosteronism, and the decrease thereof is found in decreased adrenal cortex function, primary single aldosteronism, high sodium diet, autonomic dysfunction, pregnancy hypertension syndrome, etc.

Cortisol (F) is a glucocorticoid, which is also the most important anti-stress hormone in the body, protecting the body from excessive stress. Cortisol increases blood glucose levels and provides the body with the energy required to evade injury, while acting in concert with insulin provides the cells with the glucose required to produce energy. High cortisol content humans are very immunocompetent because cortisol can suppress most immune cells, especially white blood cells. In summary, cortisol maintains life through two opposite but interrelated regulatory activities: firstly, the defensive mechanisms of the body are released and activated; and secondly, the same mechanism is closed and regulated to protect against cell damage or death caused by excessive reaction.

Dehydroepiandrosterone sulfate (DHEA-S) is secreted primarily by the adrenal cortex, and the ovary is also secreted in small amounts, a precursor of all sex hormones. The research shows that the DHEA-S has the effects of resisting aging, maintaining cardiovascular health, reducing blood fat, reducing blood pressure, improving the immune system function, enhancing bones, smoothing and refining skin, enhancing memory, increasing vitality and the like. DHEA-S peaks between ages 20-30 and decreases significantly with age.

Testosterone (T) is secreted mainly by the ovaries and adrenal glands, and the increased content is most pronounced in idiopathic precocious puberty (the level of which can be achieved in male children), adrenal hyperplasia (male children), adrenocortical tumors, and increased testosterone in women can cause hirsutism, acne, scanty menstruation or infertility, etc. Testosterone can be directly or firstly converted into Dihydrotestosterone (DHT) with stronger activity, and can be combined with androgen receptor of spermatogenic cells to promote sperm production, stimulate development of reproductive organs, promote appearance of male secondary sexual characteristics and maintain normal morphology. After male adult, excessive dihydrotestosterone can cause pathological phenomena such as acne and alopecia. In addition, prostatic hyperplasia in middle-aged and elderly people has a close relationship with dihydrotestosterone.

Androstenedione (AD) is an androgen with prohormone properties and is used primarily to assess adrenal, ovarian or testicular function. In men, 90% are secreted by the adrenal cortex. In females 50% of androstenedione is from the ovaries and 50% from the adrenal glands. Too high androstenedione in women may cause hyperandrogenism. The slightly lower levels of androstenedione measured in adult males in women of the same age, a significant increase may indicate adrenocortical hyperplasia and gonadal tumors.

Estrone (E1) and estradiol (E2) are two estrogens synthesized mainly by the ovaries, and their physiological effects are those of estrogens. Oestrone is lower in women of childbearing age than oestradiol, but obese men and near-withdrawal women have a higher oestrone content than oestradiol, and the E1/E2 ratio is significantly increased after withdrawal. Estrone is the main estrogen in women after menopause and is converted by adipocytes. Estradiol is the major estrogen responsible for regulating female characteristics, the maturation of accessory organs and the menstrual-ovulation cycle, promoting the production of the mammary duct system. Estradiol has an important role not only in reproductive and sexual function but also in other organs such as bones.

The detection of the steroid hormone can be helpful for diagnosing some endocrine disturbance diseases, such as congenital adrenocortical hyperplasia, aldosteronism, cushing syndrome and the like. At present, the immunological method is the most commonly used kit in the clinical laboratory in China, but has the defects of poor specificity, false positive or false negative results caused by interfering substances such as the amphotropic antibody and the like, and low throughput of the immunological method, and only one hormone can be measured at a time. The LC-MS/MS technology can detect retention time and target parent ions and fragment ions simultaneously, has higher specificity and less potential interference, and is particularly suitable for quantitative detection of complex biological matrixes. However, the existing LC-MS/MS technology still has a plurality of problems when detecting steroid hormone, the pretreatment mostly adopts a solid phase extraction or derivatization method, the operation is complex, the cost is high, and the clinical application is limited.

Disclosure of Invention

The invention aims to provide a kit for detecting 12 steroid hormones in serum by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology on the basis of the prior art.

The invention also aims to provide the application of the kit in detecting 12 steroid hormones in serum by utilizing an ultra-high performance liquid chromatography tandem mass spectrometry technology.

The technical scheme of the invention is as follows:

the 12 steroid hormones are respectively: progesterone (P4), 17 alpha-hydroxyprogesterone (17 alpha-OHP 4), corticosterone (CORT), dehydroepiandrosterone (DHEA), dehydroepiandrosterone Sulfate (DHEAs), testosterone (T), dihydrotestosterone (DHT), androstenedione (AD), estrone (E1), estradiol (E2), aldosterone (ALD), and cortisol (F);

the isotope internal standard corresponding to the 12 steroid hormone is respectively: progesterone-d 9 (P4-d 9), 17α -hydroxyprogesterone-d 8 (17α -OHP4-d 8), corticosterone-d 8 (CORT-d 8), dehydroepiandrosterone-d 2 (DHEA-d 2), dehydroepiandrosterone-d 6 (DHEAS-d 6), testosterone-d 3 (T-d 3), dihydrotestosterone-d 3 (DHT-d 3), androstenedione-13C 3 (AD-13C 3), estrone-d 4 (E1-d 4), estradiol-d 4 (E2-d 4), aldosterone-d 4 (ALD-d 4), and cortisol-d 4 (F-d 4);

the kit comprises the following reagents:

(1) Eluent:

Eluent a: contains 50-400 mu M ammonium fluoride aqueous solution; eluent B: methanol;

(2) Calibrator solution:

a mixed standard S0 solution containing 200ng/mL progesterone, 200ng/mL17 alpha-hydroxyprogesterone, 400ng/mL corticosterone, 400ng/mL dehydroepiandrosterone, 200000ng/mL dehydroepiandrosterone sulfate, 200ng/mL testosterone, 100ng/mL dihydrotestosterone, 200ng/mL androstenedione, 40ng/mL estrone, 40ng/mL estradiol, 40ng/mL aldosterone and 4000ng/mL cortisol was formulated as a blank serum matrix solution as a calibrator solution at eight different concentration points:

the concentrations of progesterone, 17 alpha-hydroxyprogesterone, testosterone and androstenedione are the same, and the eight concentrations are in sequence: 0.05ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml and 10ng/ml;

the concentrations of corticosterone and dehydroepiandrosterone are the same, and the eight concentrations are as follows: 0.1ng/ml, 0.2ng/ml, 0.4ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml and 20ng/ml;

the concentrations of the dehydroepiandrosterone sulfate are as follows: 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2500ng/ml, 5000ng/ml and 10000ng/ml;

the eight concentrations of dihydrotestosterone are in sequence: 0.025ng/ml, 0.05ng/ml, 0.1ng/ml, 0.25ng/ml, 0.5ng/ml, 1.25ng/ml, 2.5ng/ml and 5ng/ml;

The concentration of estrone, estradiol and aldosterone is in turn: 0.01ng/ml, 0.02ng/ml, 0.04ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml and 2ng/ml;

the eight concentrations of cortisol are in sequence: 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml and 200ng/ml;

(3) Mixing an internal standard working solution:

comprises 10ng/mL progesterone-d 9, 5ng/mL17 alpha-hydroxyprogesterone-d 8, 20ng/mL corticosterone-d 8, 20ng/mL dehydroepiandrosterone-d 2, 10000ng/mL dehydroepiandrosterone-d 6, 10ng/mL testosterone-d 3, 5ng/mL dihydrotestosterone-d 3, 10ng/mL androstenedione-13C 3, 1ng/mL estrone-d 4, 2ng/mL estradiol-d 4, 4ng/mL aldosterone-d 4 and 100ng/mL cortisol-d 4;

(4) Extract liquid: methyl tertiary butyl ether;

(5) And (3) a complex solution: 40% -60% of methanol aqueous solution;

(6) Quality control product:

blank serum matrix solution containing 12 steroid hormones, divided into low, medium and high concentrations, QC (L), QC (M) and QC (H), respectively, wherein:

QC (L) is diluted 2000-fold with blank serum matrix solution for the mixed standard S0 solution (1:1999);

QC (M) was diluted 400-fold (1:399) with blank serum matrix solution for the mixed standard S0 solution;

QC (H) was diluted 50-fold (1:49) with blank serum matrix solution for the mixed standard S0 solution.

In a preferred embodiment, the eluent A is an aqueous solution containing 50 to 100. Mu.M ammonium fluoride. In a more preferred embodiment, eluent A is an aqueous solution containing 50. Mu.M ammonium fluoride.

In one scheme, the blank serum matrix is 5% -20% of methanol aqueous solution; preferably 5% -15% aqueous methanol; more preferably 10% aqueous methanol.

In one scheme, the double solution is 45% -55% methanol aqueous solution; preferably 50% aqueous methanol.

The mixed standard S0 solution mentioned in the present invention was prepared as follows: preparing progesterone, 17 alpha-hydroxyprogesterone, corticosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone, androstenedione, estrone, estradiol, aldosterone and cortisol into standard mother liquor with concentration of 2mg/mL, 4mg/mL, 1mg/mL, 10mg/mL, 2mg/mL, 1mg/mL, 4mg/mL, 2mg/mL, 1mg/mL and 4mg/mL in sequence by using pure methanol;

the mother solution of each standard is prepared into a mixed standard S0 solution containing 200ng/mL progesterone, 200ng/mL17 alpha-hydroxyprogesterone, 400ng/mL corticosterone, 400ng/mL dehydroepiandrosterone, 200000ng/mL dehydroepiandrosterone sulfate, 200ng/mL testosterone, 100ng/mL dihydrotestosterone, 200ng/mL androstenedione, 40ng/mL estrone, 40ng/mL estradiol, 40ng/mL aldosterone and 4000ng/mL cortisol by using a methanol aqueous solution.

In preparing the mixed standard S0 solution, the aqueous methanol solution is 30% to 70% aqueous methanol solution, preferably 45% to 55% aqueous methanol solution, and more preferably 50% aqueous methanol solution.

The mixed internal standard working solution is prepared according to the following method: each isotope internal standard, including progesterone-d 9 (P4-d 9), 17 alpha-hydroxyprogesterone-d 8 (17 alpha-OHP 4-d 8), corticosterone-d 8 (CORT-d 8), dehydroepiandrosterone-d 2 (DHEA-d 2), dehydroepiandrosterone-d 6 (DHEAS-d 6), testosterone-d 3 (T-d 3), dihydrotestosterone-d 3 (DHT-d 3), androstenedione-13C 3 (AD-13C 3), estrone-d 4 (E1-d 4), estradiol-d 4 (E2-d 4), aldosterone-d 4 (ALD-d 4) and cortisol-d 4 (F-d 4), was weighed and dissolved completely with pure methanol to prepare the respective isotopes at concentrations of 1mg/mL, 5mg/mL, 2mg/mL, 1.1 mg/mL, 1mg/mL, 2.5mg/mL, 1mg/mL, and 1 mg/mL.

Each of the isotope internal standard mother solutions is prepared into an isotope internal standard SI solution containing 100ng/mL of progesterone-d 9 (P4-d 9), 50ng/mL of 17 alpha-hydroxyprogesterone-d 8 (17 alpha-OHP 4-d 8), 200ng/mL of corticosterone-d 8 (CORT-d 8), 200ng/mL of dehydroepiandrosterone-d 2 (DHEA-d 2), 100000ng/mL of dehydroepiandrosterone-d 6 (DHEAS-d 6), 100ng/mL of testosterone-d 3 (T-d 3), 50ng/mL of dihydrotestosterone-d 3 (DHT-d 3), 100ng/mL of androstenedione-13C 3 (AD-13C 3), 10ng/mL of estrone-d 4 (E1-d 4), 20ng/mL of estradiol-d 4 (E2-d 4), 40ng/mL of aldosterone-d 4 (ALD-d 4) and 1000ng/mL of cortisol-d 4 (F-d 4) by using aqueous methanol solutions.

100 mu L of SI solution is taken, 900 mu L of methanol aqueous solution is added, and the mixture is uniformly mixed to obtain mixed internal standard working solution.

When the internal standard working solution is mixed, the aqueous methanol solution is 30-70% aqueous methanol solution, preferably 45-55% aqueous methanol solution, and more preferably 50% aqueous methanol solution.

In a preferred embodiment, the kit for detecting 12 steroid hormones in serum by ultra performance liquid chromatography tandem mass spectrometry comprises the following reagents:

(1) Eluent:

eluent a: an aqueous solution containing 50. Mu.M ammonium fluoride; eluent B: methanol;

(2) Calibrator solution:

preparing progesterone, 17 alpha-hydroxyprogesterone, corticosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone, androstenedione, estrone, estradiol, aldosterone and cortisol into standard mother liquor with concentration of 2mg/mL, 4mg/mL, 1mg/mL, 10mg/mL, 2mg/mL, 1mg/mL, 4mg/mL, 2mg/mL, 1mg/mL and 4mg/mL in sequence by using pure methanol;

preparing a mixed standard S0 solution containing 200ng/mL progesterone, 200ng/mL17 alpha-hydroxyprogesterone, 400ng/mL corticosterone, 400ng/mL dehydroepiandrosterone, 200000ng/mL dehydroepiandrosterone sulfate, 200ng/mL testosterone, 100ng/mL dihydrotestosterone, 200ng/mL androstenedione, 40ng/mL estrone, 40ng/mL estradiol, 40ng/mL aldosterone and 4000ng/mL cortisol by using 50% methanol aqueous solution;

Preparing the mixed standard S0 solution into eight calibration material solutions with different concentration points by using 10% methanol solution;

(3) Mixing an internal standard working solution:

weighing each isotope internal standard, including progesterone-d 9, 17 alpha-hydroxyprogesterone-d 8, corticosterone-d 8, dehydroepiandrosterone-d 2, dehydroepiandrosterone-d 6, testosterone-d 3, dihydrotestosterone-d 3, androstenedione-13C 3, estrone-d 4, estradiol-d 4, aldosterone-d 4 and cortisol-d 4, respectively adding pure methanol to dissolve completely, and preparing into isotope internal standard mother liquor with the concentration of 1mg/mL, 5mg/mL, 2mg/mL, 1mg/mL, 0.1mg/mL, 1mg/mL, 2.5mg/mL, 1mg/mL and 1mg/mL in sequence;

preparing 50% methanol aqueous solution into isotope internal standard SI solution containing 100ng/mL progesterone-d 9, 50ng/mL17 alpha-hydroxyprogesterone-d 8, 200ng/mL corticosterone-d 8, 200ng/mL dehydroepiandrosterone-d 2, 100000ng/mL dehydroepiandrosterone-d 6, 100ng/mL testosterone-d 3, 50ng/mL dihydrotestosterone-d 3, 100ng/mL androstenedione-13C 3, 10ng/mL estrone-d 4, 20ng/mL estradiol-d 4, 40ng/mL aldosterone-d 4 and 1000ng/mL cortisol-d 4;

taking 100 mu L of SI solution, adding 900 mu L of 50% methanol aqueous solution, and uniformly mixing to obtain mixed internal standard working solution;

(4) Extract liquid: methyl tertiary butyl ether;

(5) And (3) a complex solution: 50% aqueous methanol;

(6) Quality control product:

10% aqueous methanol containing 12 steroid hormones at low, medium and high concentrations, QC (L), QC (M) and QC (H), respectively, wherein:

QC (L) contains: 0.1ng/mL progesterone (P4), 0.1ng/mL17 alpha-hydroxyprogesterone (17 alpha-OHP 4), 0.2ng/mL Corticosterone (CORT), 0.2ng/mL Dehydroepiandrosterone (DHEA), 100ng/mL dehydroepiandrosterone sulfate (DHEAS), 0.1ng/mL testosterone (T), 0.05ng/mL Dihydrotestosterone (DHT), 0.1ng/mL Androstenedione (AD), 0.02ng/mL estrone (E1), 0.02ng/mL estradiol (E2), 0.02ng/mL Aldosterone (ALD), and 2ng/mL cortisol (F).

The QC (M) includes: 0.5ng/mL progesterone (P4), 0.5ng/mL17 alpha-hydroxyprogesterone (17 alpha-OHP 4), 1ng/mL Corticosterone (CORT), 1ng/mL Dehydroepiandrosterone (DHEA), 500ng/mL dehydroepiandrosterone sulfate (DHEAS), 0.5ng/mL testosterone (T), 0.25ng/mL Dihydrotestosterone (DHT), 0.5ng/mL Androstenedione (AD), 0.1ng/mL estrone (E1), 0.1ng/mL estradiol (E2), 0.1ng/mL Aldosterone (ALD), and 10ng/mL cortisol (F).

The QC (H) includes: 5ng/mL progesterone (P4), 5ng/mL17 alpha-hydroxyprogesterone (17 alpha-OHP 4), 10ng/mL Corticosterone (CORT), 10ng/mL Dehydroepiandrosterone (DHEA), 5000ng/mL dehydroepiandrosterone sulfate (DHEAS), 5ng/mL testosterone (T), 2.5ng/mL Dihydrotestosterone (DHT), 5ng/mL Androstenedione (AD), 1ng/mL estrone (E1), 1ng/mL estradiol (E2), 1ng/mL Aldosterone (ALD), and 100ng/mL cortisol (F).

Specifically, the concentrations of QC (L), QC (M) and QC (H) corresponding to 12 steroid hormones in the quality control product are shown in Table 1;

TABLE 1 quality control product concentration (in ng/mL)

In a preferred embodiment, the calibrator solution is prepared as follows:

accurately weighing 3-5mg of each standard substance powder to be tested in a 5mL centrifuge tube, preparing standard substance mother liquor concentration in the following table by using pure methanol, diluting the rest standard substance mother liquor except Dehydroepiandrosterone (DHEAS) into use concentration of 10 mug/mL by using 50% methanol aqueous solution, and preparing each use concentration into mixed standard solution S0 solution (see table 2 for details), and uniformly mixing for later use.

Table 2 preparation of mixed standard solution S0

50. Mu.L of the mixed standard S0 solution was added to 950. Mu.L of 10% aqueous methanol as the first high concentration point S8, and the mixture was gradually diluted to S1 (see Table 3 for details), and the concentrations of the respective standard curve points were as shown in Table 3.

Table 3 preparation and concentration of standard curves

(Note: concentration units are ng/mL)

The concentration of the aqueous methanol solution referred to herein is generally referred to as the volume concentration.

In a preferred embodiment, the mixed internal standard working fluid is prepared as follows:

accurately weighing 3-5mg of each isotope internal standard substance in a 5mL centrifuge tube, preparing the concentrations of isotope internal standard mother solutions in the following table by using pure methanol, except dehydroepiandrosterone-d 6 (DHEAS-d 6) sulfate, diluting the rest isotope internal standard mother solutions into the use concentration of 10 mug/mL by using 50% methanol aqueous solution, preparing the use concentrations into mixed internal standard SI solution (see Table 4 for details), finally taking 100 mug of SI solution, adding 900 mug of 50% methanol aqueous solution, and uniformly mixing to obtain the mixed internal standard working solution.

Table 4 preparation of mixed internal standard SI solution

The application of the kit in detecting 12 steroid hormones in serum by utilizing an ultra-high performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.

The specific detection method comprises the following steps:

a method for detecting 12 steroid hormone in serum by using ultra-high performance liquid chromatography tandem mass spectrometry technology,

mixing a serum sample with internal standard solutions of all objects to be detected, obtaining the solutions to be detected through one-step liquid-liquid extraction, detecting 12 steroid hormones in pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating interference components in a target object to be detected and a serum matrix by utilizing ultra-high performance liquid chromatography, detecting mass-to-charge ratio (m/z) of the target object and corresponding isotope internal standard thereof by utilizing mass spectrometry, quantifying by utilizing an isotope internal standard method, and accurately calculating the content of the 12 steroid hormones, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a: contains 50-400 mu M ammonium fluoride aqueous solution; mobile phase B: methanol;

chromatographic column model: kineex XB-C18 (3.0 x 50mm,2.6 μm); (Phenomenex Co., USA)

And (3) performing gradient elution by using the mobile phase A and the mobile phase B as mixed mobile phases, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 60:40 to 2:98 at a constant speed within 0-3.0 minutes; within 3.0-3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 2:98; the volume ratio of mobile phase A to mobile phase B was graded from 2:98 at a constant rate to 60:40 in 3.5-5.0 minutes.

(2) Mass spectrometry conditions:

in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spray voltage was 3.0kV (ESI+)/2.5 kV (ESI-); the temperature of the ion source is 120 ℃; the temperature of the atomizing gas is 400 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; at the same time 12 steroid hormones and their corresponding isotopic internal standards were monitored.

In order to improve the chromatographic selectivity, it may be considered to adjust the polarity of the mobile phase. According to the invention, ammonium fluoride is added into the mobile phase A, so that ionization efficiency of certain target compounds can be effectively improved, and under the cooperation of other conditions, compared with the detection sensitivity of steroid hormones by adopting an LC-MS/MS method in the prior art, the pretreatment only needs one-step liquid-liquid extraction, is simple and quick, has low cost, and can detect 12 important steroid hormones simultaneously within 5 minutes. In a preferred embodiment, mobile phase A is an aqueous solution containing 50 to 100. Mu.M ammonium fluoride without affecting the effectiveness of the invention. In a more preferred embodiment, mobile phase A is an aqueous solution containing 50. Mu.M ammonium fluoride.

In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, good selectivity, high analysis speed, etc. The invention adopts 50-400 mu M ammonium fluoride aqueous solution and methanol as mobile phases, and the chromatographic column model: kinetex XB-C18 (3.0X10 mm,2.6 μm), under the cooperation of other conditions, endogenous substances do not interfere with the measurement of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, separation and detection can be completed within 5.0min, and the precision and the accuracy meet the requirements.

The selection of the internal standard is a very important task when using the internal standard method. The ideal internal standard should be capable of being added to the sample in accurate, known amounts and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; the internal standard must be sufficiently separated from the components of the sample under chromatographic conditions. The invention adopts progesterone-d 9 (P4-d 9), 17 alpha-hydroxyprogesterone-d 8 (17 alpha-OHP 4-d 8), corticosterone-d 8 (CORT-d 8), dehydroepiandrosterone-d 2 (DHEA-d 2), dehydroepiandrosterone-d 6 (DHEAS-d 6), testosterone-d 3 (T-d 3), dihydrotestosterone-d 3 (DHT-d 3), androstenedione-13C 3 (AD-13C 3), estrone-d 4 (E1-d 4), estradiol-d 4 (E2-d 4), aldosterone-d 4 (ALD-d 4) and cortisol-d 4 (F-d 4) as internal standards, and the deuterated internal standard and the tested object have the same retention time, chemical property and matrix effect, and good reproducibility and accuracy when measuring 12 steroid hormones in serum.

In one embodiment, the flow rate is 0.2 to 1.0mL/min, preferably 0.5mL/min.

Further, the column temperature is 40 to 60 ℃, preferably 50 ℃.

Further, the sample volume is 2 to 10. Mu.L, preferably 5. Mu.L.

In a preferred embodiment, when the ultra performance liquid chromatography tandem mass spectrometry technology is used for detecting 12 steroid hormones in serum, specific chromatographic conditions are as follows:

(1) High performance liquid chromatography conditions:

mobile phase a: water (containing 50 μm ammonium fluoride);

mobile phase B: methanol;

chromatographic column model: kineex XB-C18 (3.0 x 50mm,2.6 μm) (phenomenonex corporation, usa);

the gradient elution mode is adopted, and the table 5 is shown; the flow rate is 0.5mL/min, the column temperature is 50 ℃, and the sample injection volume is 5 mu L;

TABLE 5 gradient elution parameters for mobile phases

Time	Flow rate (mL/min)	％A	％B	Curve
					0.0	0.5	60	40	-
3.0	0.5	2	98	6
					3.5	0.5	2	98	6
5.0	0.5	60	40	1

(2) Mass spectrometry conditions:

in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spray voltage was 3.0kV (ESI+)/2.5 kV (ESI-); the temperature of the ion source is 120 ℃; the temperature of the atomizing gas is 400 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; meanwhile, 12 steroid hormones and corresponding isotope internal standards thereof are monitored, and mass spectrum acquisition parameters of each target object to be detected are shown in table 6.

TABLE 6 steroid hormone Mass Spectrometry parameters

The serum mentioned in the present invention is human or animal serum.

In one embodiment, the pretreated serum is prepared as follows: adding a mixed internal standard working solution into serum, adding an extraction solution for extraction after vortex, taking supernatant after centrifugal treatment, drying by nitrogen flow, mixing with a compound solution, and taking supernatant after centrifugal treatment again.

Preferably, the extract is methyl tert-butyl ether.

Preferably, the reconstituted solution is a 40% to 60% aqueous methanol solution, for example, a 50% aqueous methanol solution, without affecting the effectiveness of the present invention.

In a preferred embodiment, the pretreated serum is prepared as follows: 200 mu L of serum is taken in a 1.5mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, 800 mu L of methyl tertiary butyl ether is added into the centrifuge tube for extraction after vortex, 700 mu L of supernatant is taken after centrifugation at 15000r/min for 5min at 15 ℃, 80 mu L of 50% methanol aqueous solution is mixed after drying by nitrogen flow, and 60 mu L of supernatant is taken after centrifugation at 15000r/min for 3 min.

In a more preferred embodiment, the pretreated serum is prepared as follows: 200 mu L of serum is taken in a 1.5mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, and then vortex is carried out for 5s; then 800 mu L of methyl tertiary butyl ether is added, and high-speed oscillation (maximum vibration speed) is carried out for 5min; centrifuging at 15 ℃ for 5min at a rotating speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen to near dryness (without heating); 80. Mu.L of 50% methanol (covered with a lid) was added, the mixture was centrifuged at a high speed for 3min with shaking at a high speed for 2min at 15000r/min, 60. Mu.L of the supernatant was removed from the liner tube and the sample was sampled at a sample loading rate of 5. Mu.L.

In one scheme, the mixed internal standard working solution is prepared according to the following method:

weighing each isotope internal standard, including progesterone-d 9 (P4-d 9), 17 alpha-hydroxyprogesterone-d 8 (17 alpha-OHP 4-d 8), corticosterone-d 8 (CORT-d 8), dehydroepiandrosterone-d 2 (DHEA-d 2), dehydroepiandrosterone-d 6 (DHEAS-d 6), testosterone-d 3 (T-d 3), dihydrotestosterone-d 3 (DHT-d 3), androstenedione-13C 3 (AD-13C 3), estrone-d 4 (E1-d 4), estradiol-d 4 (E2-d 4), aldosterone-d 4 (ALD-d 4) and cortisol-d 4 (F-d 4), and adding pure methanol to dissolve completely to prepare the isotopes in the concentrations of 1mg/mL, 5mg/mL, 2mg/mL, 1.1 mg/mL, 1mg/mL, 2.5mg/mL, 1mg/mL and 1mg/mL respectively;

the isotopic internal standard mother liquor is prepared into an isotopic internal standard SI solution containing 100ng/mL of progesterone-d 9 (P4-d 9), 50ng/mL of 17 alpha-hydroxyprogesterone-d 8 (17 alpha-OHP 4-d 8), 200ng/mL of corticosterone-d 8 (CORT-d 8), 200ng/mL of dehydroepiandrosterone-d 2 (DHEA-d 2), 100000ng/mL of dehydroepiandrosterone-d 6 sulfate (DHEAS-d 6), 100ng/mL of testosterone-d 3 (T-d 3), 50ng/mL of dihydrotestosterone-d 3 (DHT-d 3), 100ng/mL of androstenedione-13C 3 (AD-13C 3), 10ng/mL of estrone-d 4 (E1-d 4), 20ng/mL of estradiol-d 4 (E2-d 4), 40ng/mL of aldosterone-d 4 (ALD-d 4) and 1000ng/mL of cortisol-d 4 (F-d 4) by 50% aqueous methanol solution;

100 mu L of SI solution is taken, 900 mu L of 50% methanol aqueous solution is added, and the mixture is uniformly mixed to obtain mixed internal standard working solution.

In one embodiment, the standard solution is prepared as follows:

progesterone (P4), 17 alpha-hydroxyprogesterone (17 alpha-OHP 4), corticosterone (CORT), dehydroepiandrosterone (DHEA), dehydroepiandrosterone (DHEAS), testosterone (T), dihydrotestosterone (DHT), androstenedione (AD), estrone (E1), estradiol (E2), aldosterone (ALD) and cortisol (F) were formulated in pure methanol at standard stock solutions of concentrations of 2mg/mL, 4mg/mL, 1mg/mL, 10mg/mL, 2mg/mL, 1mg/mL, 4mg/mL, 2mg/mL, 1mg/mL and 4mg/mL in this order.

Each of the above standard stock solutions was then formulated with 50% aqueous methanol as a mixed standard S0 solution containing 200ng/mL progesterone (P4), 200ng/mL17 alpha-hydroxyprogesterone (17 alpha-OHP 4), 400ng/mL Corticosterone (CORT), 400ng/mL Dehydroepiandrosterone (DHEA), 200000ng/mL dehydroepiandrosterone sulfate (DHEAS), 200ng/mL testosterone (T), 100ng/mL Dihydrotestosterone (DHT), 200ng/mL Androstenedione (AD), 40ng/mL estrone (E1), 40ng/mL estradiol (E2), 40ng/mL Aldosterone (ALD), and 4000ng/mL cortisol (F).

Preparing eight calibrator solutions with different concentration points from the mixed standard S0 solution by using a blank serum matrix (10% methanol aqueous solution), wherein the eight concentration points of the calibrator solutions are as follows:

The concentrations of progesterone (P4), 17 a-hydroxyprogesterone (17 a-OHP 4), testosterone (T) and Androstenedione (AD) are the same, with eight concentrations in sequence: 0.05ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml and 10ng/ml;

the concentrations of Corticosterone (CORT) and Dehydroepiandrosterone (DHEA) are identical, with eight concentrations in order: 0.1ng/ml, 0.2ng/ml, 0.4ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml and 20ng/ml;

the eight concentrations of Dehydroepiandrosterone (DHEAS) were in order: 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2500ng/ml, 5000ng/ml and 10000ng/ml;

the eight concentrations of Dihydrotestosterone (DHT) are in order: 0.025ng/ml, 0.05ng/ml, 0.1ng/ml, 0.25ng/ml, 0.5ng/ml, 1.25ng/ml, 2.5ng/ml and 5ng/ml;

the eight concentrations of estrone (E1), estradiol (E2) and Aldosterone (ALD) are in order: 0.01ng/ml, 0.02ng/ml, 0.04ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml and 2ng/ml;

the eight concentrations of cortisol (F) are in order: 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml and 200ng/ml.

200 mu L of each concentration point sample is taken, 20 mu L of mixed internal standard working solution is added into the sample, and then vortex is carried out for 5s; then 800 mu L of methyl tertiary butyl ether is added, and high-speed oscillation (maximum vibration speed) is carried out for 5min; centrifuging at 15 ℃ for 5min at a rotating speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen to near dryness (without heating); 80. Mu.L of 50% methanol (covered with a lid) was added, the mixture was centrifuged at a high speed for 3min with shaking at a high speed for 2min at 15000r/min, 60. Mu.L of the supernatant was removed from the liner tube and the sample was sampled at a sample loading rate of 5. Mu.L.

In one scheme, the quality control product is prepared according to the following method:

the mixed standard S0 solution is taken to prepare three different concentrations of QC (L), QC (M) and QC (H) by using a blank serum matrix (10% methanol aqueous solution).

Respectively taking 200 mu L of quality control product solution QC (L), QC (M) and QC (H) in a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard working solution into the centrifuge tube, and then swirling for 5s; then 800 mu L of methyl tertiary butyl ether is added, and high-speed oscillation (maximum vibration speed) is carried out for 5min; centrifuging at 15 ℃ for 5min at a rotating speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen to near dryness (without heating); 80. Mu.L of 50% methanol (covered with a lid) was added, the mixture was centrifuged at a high speed for 3min with shaking at a high speed for 2min at 15000r/min, 60. Mu.L of the supernatant was removed from the liner tube and the sample was sampled at a sample loading rate of 5. Mu.L.

By adopting the technical scheme of the invention, the advantages are as follows:

the kit provided by the invention can be used for detecting 12 steroid hormones in serum, a serum sample is mixed with the mixed internal standard solution of all the objects to be detected, the sample does not need derivatization treatment, and the solution to be detected can be obtained only through one-step liquid-liquid extraction; by adding electrolyte ammonium fluoride in the mobile phase, the ionization efficiency of certain target compounds can be effectively improved; and then 12 hormones are detected simultaneously by adopting a mass spectrum positive-negative switching scanning mode, so that the method has the advantages of high sensitivity, strong specificity, low cost and simple pretreatment process, can finish the separation and detection of the 12 hormones within 5.0min, and provides a reliable detection method for the health evaluation of clinical endocrine diseases.

Drawings

FIG. 1 is a selective ion flow chromatogram of a 12 steroid hormone standard;

FIG. 2 is a selective ion flow chromatogram of 12 steroid hormones in a serum sample.

Detailed Description

The kits of the present invention are further illustrated by the following examples, which are not intended to limit the invention in any way.

Example 1:

1. experimental materials and instruments

1. Material

The samples were from serum samples collected from the 3 month clinic in 2018 of the wuhan asian heart disease hospital.

(1) Instrument: a Xex TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra-high performance liquid chromatography system (auto sampler, waters Corporation); SCILOGEX D2012 high speed table centrifuge (usa); ultrapure water meter (ELGA LabWater, uk); multitube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L); glassware, measuring cylinders, etc.

(2) Reagent consumable: MS grade methanol (Fisher, USA); HPLC grade methanol (Honeywell, usa); HPLC grade methyl tert-butyl ether (Fisher, usa); column Kinetex XB-C18 (3.0X105 mm,2.6 μm) (Phenomenex Co., U.S.A.).

(3) Standard substance: p4, 17α -OHP4, CORT, T, DHT, AD, E and F were purchased from Dr. Ehrensorfer, germany; ALD, ALD-d4, P4-d9, CORT-d8, 17α -OHP4-d8, DHEA-d2, F-d4, E1-d4, E2-d4, available from TRC; DHEA and T-d3 are available from Cerelliant; DHEAS, DHEAS-d6 and AD-13C3 are purchased from IsoSciences; e2 and DHT-d3 were purchased from Sigma.

(4) Quality control product: the quality control product contains QC (L), QC (M) and QC (H) concentrations corresponding to 12 steroid hormones shown in Table 1.

2. Liquid condition

(1) Chromatographic conditions: mobile phase a: water (containing 50 μm ammonium fluoride-water solution); mobile phase B: methanol. Chromatographic column model: kinetex XB-C18 (3.0X10 mm,2.6 μm) was eluted by gradient, as detailed in Table 5. The flow rate was 0.5mL/min, the column temperature was 50deg.C, and the sample volume was 5. Mu.L.

(2) Mass spectrometry conditions: in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spray voltage was 3.0kV (ESI+)/2.5 kV (ESI-); the temperature of the ion source is 120 ℃; the temperature of the atomizing gas is 400 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; meanwhile, 12 steroid hormones and corresponding isotope internal standards thereof are monitored, and mass spectrum acquisition parameters of each target object to be detected are shown in table 6.

2. Experimental procedure

(1) Preparing a standard substance:

accurately weighing 3-5mg of each standard in a 5mL centrifuge tube (3 mg of standard below standard does not need to be weighed and is completely dissolved), preparing standard mother liquor concentration by using pure methanol, diluting 11 standard mother liquor into use concentration of 10 mug/mL by using 50% methanol aqueous solution (DHEAS use concentration is standard mother liquor concentration 10000 mug/mL), and preparing each use concentration into mixed standard S0 solution (see table 2 for details).

The mixed standard S0 solution is prepared into eight calibration material solutions with different concentration points by a blank serum matrix (10% methanol aqueous solution), and the preparation process is as follows:

(2) Preparation of mixed internal standard working solution

Accurately weighing 3-5mg of each isotope internal standard substance in a 5mL centrifuge tube (standard substances with the specification below 3mg do not need to be weighed and are completely dissolved), preparing the isotope internal standard mother liquor concentration by using pure methanol, diluting the 11 isotope internal standard mother liquor into the use concentration of 10 mug/mL by using 50% methanol aqueous solution (DHEAS-d 6 use concentration is the isotope internal standard mother liquor concentration of 1000 mug/mL), preparing the use concentrations into mixed internal standard SI solution (see table 4 for details), finally taking 100 mug of SI solution, adding 900 mug of 50% methanol, and uniformly mixing to obtain mixed internal standard working solution.

(3) Preparing a quality control product:

the mixed standard S0 solution is prepared into three different concentrations of QC (L), QC (M) and QC (H) by 10% methanol aqueous solution.

(4) Sample processing

1) Pretreatment of standard products: 200 mu L of each concentration point sample is taken, 20 mu L of mixed internal standard working solution is added into the sample, and then vortex is carried out for 5s; then 800 mu L of methyl tertiary butyl ether is added, and high-speed oscillation (maximum vibration speed) is carried out for 5min; centrifuging at 15 ℃ for 5min at a rotating speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen to near dryness (without heating); 80. Mu.L of 50% methanol (covered with a lid) was added, the mixture was centrifuged at a high speed for 3min with shaking at a high speed for 2min at 15000r/min, 60. Mu.L of the supernatant was removed from the liner tube and the sample was sampled at a sample loading rate of 5. Mu.L.

2) Pretreatment of serum samples: 200 mu L of serum is taken in a 1.5mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, and then vortex is carried out for 5s; then 800 mu L of methyl tertiary butyl ether is added, and high-speed oscillation (maximum vibration speed) is carried out for 5min; centrifuging at 15 ℃ for 5min at a rotating speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen to near dryness (without heating); 80. Mu.L of 50% methanol (covered with a lid) was added, the mixture was centrifuged at a high speed for 3min with shaking at a high speed for 2min at 15000r/min, 60. Mu.L of the supernatant was removed from the liner tube and the sample was sampled at a sample loading rate of 5. Mu.L.

3) Pretreatment of quality control products: 200. Mu.L of each of quality control solution QC (L), QC (M) and QC (H) was taken in a 1.5mL centrifuge tube and then was consistent with pretreatment of serum samples, which will not be described in detail herein.

Adding films on the upper and lower periphery of the kit, carrying out shock prevention and heat preservation, placing eluent A and eluent B on the upper left, and respectively placing 12 ampoule bottles on the lower left, wherein the ampoule bottles are respectively a calibrator, a mixed internal standard working solution and a quality control product; on the right, 10mL of the reconstituted solution and 120mL of the extract were placed, respectively.

The components of the assay kit are shown in Table 7.

Table 7 vitamin analysis kit Components (100 parts)

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4. Method verification

In the detection method, the peak shape of the 12 steroid hormone standard is symmetrical to that of a serum sample, and no impurity peak interference exists, so that good detection can be achieved under the condition, and FIG. 1 is a selective ion flow chromatogram of the 12 steroid hormone standard; FIG. 2 is a selective ion flow chromatogram of 12 steroid hormones in a serum sample.

1. Standard curve:

and (3) using an isotope internal calibration method, using TargetLynx software to establish a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of a steroid hormone to be detected in serum. The linear fitting equation of 12 steroid hormone in the respective concentration range has good linearity, and the correlation coefficient is more than 0.997, and is shown in Table 8 in detail.

TABLE 8 retention time and linear range of steroid hormones

2. Minimum limit of quantification

The minimum quantitative limit (LLOQ) is taken as the lowest point of the curvilinearity range, and also reflects the sensitivity of the method. Because steroid hormones have low content in human bodies and similar structures, the sensitivity and specificity requirements of the method are very high, the inventor optimizes and surveys the detection method, and the minimum quantitative limit (LLOQ) at present basically meets the sensitivity requirements of simultaneous detection of 12 male and female sex hormones, and the concentration of the LLOQ is shown in Table 9.

Table 9 method quantitative lower limit data table

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3. Accuracy investigation: and (5) evaluating the accuracy of the method by adopting a standard adding recovery rate test. One example of human serum samples was randomly selected, 1 of which was not added with standard, 2 other samples were added with low and high 2 concentrations of standard, and the same procedure was repeated and measured 5 times to calculate recovery results, as shown in Table 10. The results show that the standard recovery rate of 12 steroid hormones in serum is between 85% and 115%, and all the results meet the requirements.

Table 10 results of the labeled recovery of 12 steroid hormone in serum (Unit ng/mL)

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4. Precision test: the serum samples of normal persons were taken and treated repeatedly for 6 batches in one day for 3 days, the concentration of 12 steroid hormones was quantitatively determined by the isotope internal standard method, the precision in and among the batches was counted for three consecutive days, and the calculation results are shown in Table 11.

TABLE 11 results of within-batch and inter-batch precision tests (Unit ng/mL)

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5. Discussion of the invention

The invention adopts UPLC-MS/MS method to measure 12 steroid hormones of progesterone (P4), 17 alpha-hydroxyprogesterone (17 alpha-OHP 4), corticosterone (CORT), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone (T), dihydrotestosterone (DHT), androstenedione (AD), estrone (E1), estradiol (E2), aldosterone (ALD) and cortisol (F) in human serum. At present, the pretreatment mostly adopts a solid-phase extraction or derivatization method, the operation is complex, the cost is high, and the clinical application is limited. Mixing a serum sample with internal standard solutions of all objects to be detected, and obtaining a solution to be detected through one-step liquid-liquid extraction; by adding electrolyte ammonium fluoride in the mobile phase, the ionization efficiency of certain target compounds is improved; the mass spectrum positive-negative switching scanning mode is adopted to detect 12 hormones simultaneously.

The invention examines the precision and the standard recovery rate of 12 steroid hormone in serum between batches, the results are between 85% and 115%, and the requirements are met; meanwhile, because the content of hormone in the human body is extremely low, the requirement on sensitivity is very high, and the minimum quantitative limit of the method can reach 10pg/mL, so that the clinical detection requirement is basically met.

The method has the advantages of high sensitivity, strong specificity, low cost and simple pretreatment process, can finish the separation and detection of 12 hormones within 5.0min, and provides a reliable detection method for the health evaluation of clinical endocrine diseases.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An application of a kit in detecting 12 steroid hormone in serum by utilizing an ultra-high performance liquid chromatography tandem mass spectrometry technology,

the 12 steroid hormones are respectively: progesterone, 17 a-hydroxyprogesterone, corticosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone, androstenedione, estrone, estradiol, aldosterone, and cortisol;

the isotope internal standard corresponding to the 12 steroid hormone is respectively: progesterone-d 9, 17 a-hydroxyprogesterone-d 8, corticosterone-d 8, dehydroepiandrosterone-d 2, dehydroepiandrosterone-d 6, testosterone-d 3, dihydrotestosterone-d 3, androstenedione-13C 3, estrone-d 4, estradiol-d 4, aldosterone-d 4, and cortisol-d 4;

The kit comprises the following reagents:

(1) Eluent:

(2) Calibrator solution:

progesterone, 17 a-hydroxyprogesterone, corticosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone, androstenedione, estrone, estradiol, aldosterone, and cortisol are formulated in pure methanol to give standard stock solutions at concentrations of 2 mg/mL, 2 mg/mL, 4 mg/mL, 1mg/mL, 10 mg/mL, 2 mg/mL, 1mg/mL, 4 mg/mL, 2 mg/mL, 1mg/mL, 1mg/mL, and 4 mg/mL, in that order;

preparing 50% methanol aqueous solution into mixed standard S0 solution containing 200ng/mL progesterone, 200ng/mL17 a-hydroxyprogesterone, 400 ng/mL corticosterone, 400 ng/mL dehydroepiandrosterone, 200000 ng/mL dehydroepiandrosterone sulfate, 200ng/mL testosterone, 100ng/mL dihydrotestosterone, 200ng/mL androstenedione, 40ng/mL estrone, 40ng/mL estradiol, 40ng/mL aldosterone and 4000ng/mL cortisol;

preparing the mixed standard S0 solution into eight calibration material solutions with different concentration points by using 10% methanol solution; the eight concentration points of the calibrator solution are:

the concentrations of progesterone, 17 a-hydroxyprogesterone, testosterone and androstenedione are the same, and the eight concentrations are in sequence: 0.05ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml and 10ng/ml;

(3) Mixing an internal standard working solution:

weighing each isotope internal standard, including progesterone-d 9, 17 a-hydroxyprogesterone-d 8, corticosterone-d 8, dehydroepiandrosterone-d 2, dehydroepiandrosterone-d 6, testosterone-d 3, dihydrotestosterone-d 3, androstenedione-13C 3, estrone-d 4, estradiol-d 4, aldosterone-d 4 and cortisol-d 4, respectively adding pure methanol to dissolve completely, and preparing into isotope internal standard mother liquor with the concentration of 1 mg/mL, 1 mg/mL, 5 mg/mL, 2 mg/mL, 1 mg/mL, 0.1 mg/mL, 1 mg/mL, 1 mg/mL, 2.5 mg/mL, 1 mg/mL, 1 mg/mL and 1 mg/mL in sequence;

Preparing the isotope internal standard mother solution into an isotope internal standard SI solution containing 100 ng/mL progesterone-d 9, 50 ng/mL17 a-hydroxyprogesterone-d 8, 200 ng/mL corticosterone-d 8, 200 ng/mL dehydroepiandrosterone-d 2, 100000 ng/mL dehydroepiandrosterone-d 6, 100 ng/mL testosterone-d 3, 50 ng/mL dihydrotestosterone-d 3, 100 ng/mL androstenedione-13C 3, 10ng/mL estrone-d 4, 20 ng/mL estradiol-d 4, 40 ng/mL aldosterone-d 4 and 1000 ng/mL cortisol-d 4 by using 50% methanol aqueous solution;

(4) Extract liquid: methyl tertiary butyl ether;

(5) And (3) a complex solution: 50% aqueous methanol;

(6) Quality control product:

10% aqueous methanol containing 12 steroid hormones at low, medium and high concentrations of L-QC, M-QC and H-QC, respectively, wherein:

the L-QC comprises: 0.1ng/mL progesterone, 0.1ng/mL17 a-hydroxyprogesterone, 0.2ng/mL corticosterone, 0.2ng/mL dehydroepiandrosterone, 100 ng/mL dehydroepiandrosterone sulfate, 0.1ng/mL testosterone, 0.05ng/mL dihydrotestosterone, 0.1ng/mL androstenedione, 0.02ng/mL estrone, 0.02ng/mL estradiol, 0.02ng/mL aldosterone, and 2ng/mL cortisol;

the M-QC comprises: 0.5ng/mL progesterone, 0.5ng/mL17 a-hydroxyprogesterone, 1ng/mL corticosterone, 1ng/mL dehydroepiandrosterone, 500 ng/mL dehydroepiandrosterone sulfate, 0.5ng/mL testosterone, 0.25ng/mL dihydrotestosterone, 0.5ng/mL androstenedione, 0.1ng/mL estrone, 0.1ng/mL estradiol, 0.1ng/mL aldosterone, and 10ng/mL cortisol;

The H-QC comprises: 5ng/mL progesterone, 5ng/mL17 a-hydroxyprogesterone, 10ng/mL corticosterone, 10ng/mL dehydroepiandrosterone, 5000 ng/mL dehydroepiandrosterone sulfate, 5ng/mL testosterone, 2.5ng/mL dihydrotestosterone, 5ng/mL androstenedione, 1ng/mL estrone, 1ng/mL estradiol, 1ng/mL aldosterone, and 100ng/mL cortisol;

the method comprises the steps of detecting 12 steroid hormones in pretreated serum by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry technology, separating an object to be detected from interfering components in serum matrixes by utilizing the ultra-high performance liquid chromatography, detecting mass-charge ratios of the object to be detected and corresponding isotope internal standards, quantifying by utilizing an isotope internal standard method, and accurately calculating the content of the 12 steroid hormones, wherein specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a: an aqueous solution containing 50. Mu.M ammonium fluoride; mobile phase B: methanol;

chromatographic column model: kineex XB-C18.0 x 50 mm,2.6 μm;

and (3) performing gradient elution by using the mobile phase A and the mobile phase B as mixed mobile phases, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 60:40 to 2:98 at a constant speed within 0-3.0 minutes; within 3.0-3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 2:98; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 60:40 at a constant speed within 3.5-5.0 minutes; the flow rate is 0.5 mL/min; column temperature is 50 ℃; the sample injection volume is 5 mu L;

(2) Mass spectrometry conditions:

in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spraying voltage is 3.0 kV ESI+/2.5kV ESI-; the temperature of the ion source is 120 ℃; the temperature of the atomizing gas is 400 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; simultaneously, 12 steroid hormones and corresponding isotope internal standards thereof are monitored;

the pretreated serum was prepared as follows: 200 mu L of serum is taken in a 1.5 mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, 800 mu L of methyl tertiary butyl ether is added into the centrifuge tube for extraction after vortex, 700 mu L of supernatant is taken after centrifugation at 15 ℃ for 5min at 15000 r/min, the supernatant is dried by nitrogen flow and then mixed with 80 mu L of 50% methanol aqueous solution, and 60 mu L of supernatant is taken after centrifugation at 15000 r/min for 3 min;

pretreatment of the calibrator was prepared as follows: 200 mu L of each concentration point sample is taken, 20 mu L of mixed internal standard working solution is added into the sample, and then vortex is carried out for 5s; adding 800 mu L of methyl tertiary butyl ether, and oscillating for 5min at a high speed; centrifuging at 15 ℃ for 5min at a rotation speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen until the supernatant is nearly dry; adding 80 mu L of 50% methanol aqueous solution, oscillating at a high speed for 2 min, centrifuging at 15000 r/min for 3min, transferring 60 mu L of supernatant into a lining pipe for sample loading detection, and sampling the sample with a sample feeding amount of 5 mu L;

The pretreatment of the quality control product is prepared according to the following method: respectively taking 200 mu L of quality control product solution L-QC, 200 mu L of M-QC and 200 mu L of H-QC in a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution into the centrifuge tubes, and then swirling for 5s; adding 800 mu L of methyl tertiary butyl ether, and oscillating for 5min at a high speed; centrifuging at 15 ℃ for 5min at a rotation speed of 15000 r/min; transferring 700 mu L of supernatant into another centrifuge tube, and blowing nitrogen until the supernatant is nearly dry; adding 80 mu L of 50% methanol aqueous solution, oscillating at a high speed for 2 min, centrifuging at 15000 r/min for 3 min, transferring 60 mu L of supernatant into the lining pipe for sample loading detection, and sampling the sample with a sample feeding amount of 5 mu L.