A method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones

Abstract

The present invention discloses a method for simultaneously detecting a plurality of fat-soluble vitamins and steroid hormones, comprising the following steps: adding an internal standard to a serum sample, mixing, and then adding methyl tert-butyl ether for extraction; extracting the components to be tested into an organic solvent, centrifuging, drying the extract with nitrogen, re-dissolving, and then injecting and analyzing the extract on a liquid chromatography-mass spectrometry system. The detection method of the present invention can simultaneously detect the contents of five fat-soluble vitamins and seventeen steroid hormones in serum (plasma), and simultaneously test a total of 22 substances to be tested, which is accurate and can be quantitatively detected. The present invention adopts a scheme that changes the conventional prior art of using the isotope of the substance to be tested as the internal standard substance, and adopts trenbolone cyclohexylmethyl carbonate as the internal standard, which can simultaneously quantify 22 substances to be tested, greatly reducing the workload.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones. Background Art

[0002] Fat-soluble vitamins are a group of vitamins containing a ring structure and a long aliphatic hydrocarbon chain. They are insoluble in water but soluble in fat and organic solvents. They mainly include vitamin A, vitamin D, vitamin E, and vitamin K. Vitamin A can maintain normal vision, prevent night blindness, promote growth and development, and treat dry eye disease. Vitamin A is commonly found in carrots, animal livers, vegetables, and fruits. Vitamin D regulates the body's calcium and phosphorus metabolism and promotes bone growth and development. Vitamin D is commonly found in animal livers, fish, and dairy products. Vitamin E helps delay aging and is anti-oxidant. It is commonly found in edible oils, fruits, vegetables, and grains. Vitamin K is a substance necessary for maintaining blood coagulation. If the body lacks vitamin K, it often has a tendency to bleed. Vitamin K is commonly found in onions, green vegetables, dairy products, etc.

[0003] Steroid hormones are a large class of hormones, mainly including glucocorticoids, mineralocorticoids and gonadal hormones, all of which are secreted by different levels of the adrenal cortex. There are many steroid hormones in the body, each of which plays a different role. For example: Glucocorticoids: mainly including cortisone and prednisone, which are commonly used in clinical practice, have anti-inflammatory, anti-allergic and blood sugar-raising effects. When glucocorticoids are used excessively, moon face, buffalo hump and centripetal obesity are prone to occur; Mineralocorticoids: can regulate the body's water and salt metabolism. If mineralocorticoids are excessive, patients are prone to blood pressure fluctuations; Gonadal hormones: gonadal hormones mainly include progesterone, estrogen, testosterone, and androgen, which can promote the development of sexual organs and maintain secondary sexual characteristics. If the level of gonadal hormones is abnormal, it is easy to cause menstrual disorders and infertility; Other hormones: 1,25-dihydroxyvitamin D3 is a type of hormone that helps calcium absorption. It also belongs to steroid hormones and is mainly used for the regulation of calcium and phosphorus. If the synthesis of 1,25-dihydroxyvitamin D3 is insufficient, osteoporosis, rickets, etc. are prone to occur. In addition, there are many types of steroid hormones in the body. Once an abnormality occurs, you should go to the hospital for treatment in time. Steroid hormones play an important role in the body. If the level of steroid hormones is too low, or if the cholesterol content in the body is too low, it will affect the synthesis and secretion of steroid hormones. The hormone level metabolism in the body is abnormal, and the body will be in a pathological state.

[0004] After searching: Chinese patent application CN110133135A discloses a method for determining the content of multiple fat-soluble vitamins. The serum sample is detected by liquid phase mass spectrometry, and the content of multiple fat-soluble vitamins in the serum sample is obtained by inserting the standard curve, with the isotope as the internal standard. Compared with the existing detection methods, the method of the invention can detect multiple fat-soluble vitamins at the same time.

[0005] Chinese patent application CN114689740A discloses a method for simultaneously determining six kinds of CAH related steroid hormones. The six kinds of related steroid hormones are 17α-hydroxyprogesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol, cortisol and corticosterone; the determination method comprises the following steps: step S1: prepare mixed internal standard, quality control and standard curve products respectively, and use isotope as internal standard substance; step S2: prepare sample extract; step S3: extract and incubate sample; step S4: concentrate and re-dissolve sample; step S5: measure the re-dissolved solution with liquid chromatography tandem mass spectrometer, draw calibration curve, and calculate the concentration of 17α-hydroxyprogesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol, cortisol and corticosterone in the sample to be tested. This method can simultaneously determine multiple steroid hormones.

[0006] At present, the methods for determining these two types of substances include liquid chromatography and liquid chromatography-mass spectrometry. The number of fat-soluble vitamins or steroid hormones that can be determined simultaneously does not exceed six, and the internal standards are all isotopes of the target substances, so many internal standards need to be prepared for quantitative analysis. The above schemes not only require a large amount of sample, but are also time-consuming and labor-intensive, and have their limitations when measuring small sample volumes and many items. Summary of the invention

[0007] In order to make up for the shortcomings of the existing methods, the present invention provides a method for determining the contents of multiple steroid hormones and multiple fat-soluble vitamins in serum (plasma) with high selectivity and accurate quantification.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] A method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones comprises the following steps:

[0010] Add the internal standard to the serum sample, mix well, and then add the extractant for extraction; extract the component to be tested into the organic solvent, centrifuge, transfer the extract to another centrifuge tube, blow dry, re-dissolve, and then inject and analyze on the liquid chromatography-mass spectrometry system; prepare the standard solution at the same time;

[0011] Liquid chromatography conditions:

[0012] Mobile phase A: 0.1-0.5% formic acid solution; Mobile phase B: methanol solution containing 0.1-0.5% formic acid; Chromatographic column model: C18, 2.0×150mm, 3.0μm; Flow rate: 0.3-0.4ml / min; Column temperature: 40℃-45℃;

[0013] Gradient elution was used, and the gradient elution program was as follows: 0-0.5min, the volume percentage of mobile phase B was 45%; 0.5-3min, the volume percentage of mobile phase B gradually decreased to 90%; 3-5min, the volume percentage of mobile phase B was 90%, and from 5-5.1min, the volume percentage of mobile phase B decreased to 45%; 5.1-5.5min, the volume percentage of mobile phase B was 45%;

[0014] Mass spectrometry conditions: atmospheric pressure chemical ionization source positive ion mode, multiple reaction monitoring; ion source voltage was 4 kV (APCI+); ion transfer tube temperature was 340°C-360°C; nebulizer gas temperature was 340°C-360°C, and sheath gas flow rate was 40-50 L / min.

[0015] Preferably, the internal standard is trenbolone cyclohexyl methyl carbonate at a concentration of 0.8-1.2 μg / ml, preferably 1 μg / ml. The internal standard of the present invention: trenbolone cyclohexyl methyl carbonate is a chemical substance with a chemical structure similar to that of the substance to be measured, and the chemical formula is C 26 H 34 O 4 , CAS No. 23454-33-3, molecular weight 410.55. Density 1.17g / cm 3 , boiling point 607.9℃ at760mmHg, flash point 261.7℃.

[0016] Preferably, the extractant is methyl tert-butyl ether, and the ratio of the volume of the extractant to the volume of the sample is (5-10):1.

[0017] Preferably, the extract is blown dry using nitrogen.

[0018] Preferably, the re-dissolving solvent is methanol.

[0019] Preferably, the liquid chromatography detection conditions are: Athena C18-WP, 2.1×150 mm, 3 μm; flow rate: 0.4 ml / min; column temperature: 45° C.; mobile phase: A: 0.1% formic acid water; B: methanol containing 0.1% formic acid.

[0020] The percentage content of the present invention is the mass percentage content.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] 1. The detection method of the present invention can simultaneously detect the contents of five steroid hormones and seventeen fat-soluble vitamins in serum (plasma), and a total of 22 substances to be tested are tested simultaneously, which is accurate and can be quantitatively detected.

[0023] 2. The present invention changes the conventional prior art of using the isotope of the test substance as the internal standard substance and adopts trenbolone cyclohexylmethyl carbonate as the internal standard, which can quantify 23 test substances at the same time, greatly reducing the workload. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the total ion map (TIC);

[0025] Figure 2 is the extracted ion map of VitA;

[0026] Figure 3 is the extracted ion map of VitE;

[0027] Figure 4 is the extracted ion map of VitK1;

[0028] Figure 5 is the extracted ion map of 25-OHVitD2;

[0029] Figure 6 is the extracted ion map of 25-OHVitD3;

[0030] Figure 7 is the extracted ion map of E1;

[0031] Figure 8 is the extracted ion map of E2;

[0032] Fig. 9 is the extracted ion map of E3;

[0033] Fig.10 is the extracted ion map of A4;

[0034] Fig.11 is the extracted ion map of P;

[0035] Fig.12 is the extracted ion map of TESTO;

[0036] Fig.13 is the extracted ion map of DHT;

[0037] Fig.14 is the extracted ion map of PREG;

[0038] Fig.15 is the extracted ion map of HCOR;

[0039] Fig.16 is the extracted ion map of DHEA;

[0040] Fig.17 is the extracted ion map of ALD;

[0041] Fig.18 is the extracted ion map of CORTISONE;

[0042] Fig.19 This is the extracted ion map of 11DOCOR;

[0043] Fig. 20 is the extracted ion diagram of 17-OHP;

[0044] Fig.21 is the extracted ion map of 17-OHPREG;

[0045] Fig. 22 This is the extracted ion map of 21DOCOR;

[0046] Fig.23 is the extracted ion map of 21OHPREG;

[0047] Fig.24 This is the extracted ion map of TCMHC. DETAILED DESCRIPTION

[0048] The detailed structure of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0049] Example 1

[0050] 1. Reagents (all of which are available on the market): testosterone, estrone, progesterone, estradiol, estriol, androstenedione, dihydrotestosterone, pregnenolone, 17-hydroxyprogesterone (17α-hydroxyprogesterone), dehydroepiandrosterone, 17α-hydroxypregnenolone, 21-hydroxypregnenolone, cortisol, corticosterone, aldosterone, deoxycorticosterone, 11-deoxycortisol, 18-hydroxycortisol, 21-deoxycortisol, vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1. The internal standard is trenbolone cyclohexylmethyl carbonate.

[0051] Methanol, acetonitrile, methyl tert-butyl ether, formic acid, artificial serum.

[0052] 2. Mobile phase A: 0.1-0.5% formic acid in water; Mobile phase B: methanol (containing 0.1-0.5% formic acid);

[0053] Preparation of standard solution: Accurately weigh 10 mg of each standard into a 10 mL volumetric flask, dissolve it with methanol, and add to the scale to make a concentration of 1 mg / mL. Store at -80 °C. When used, dilute with methanol to the working solution concentration.

[0054] 3. Preparation of working curve: Take the standard solution mixture and dilute it with artificial serum to the following concentration gradient as shown in Table 1:

[0055] Table 1 Artificial serum dilution concentration gradient table

[0056]

[0057]

[0058] 4. Instruments: ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, high-speed refrigerated centrifuge, mixer, nitrogen blower, analytical balance.

[0059] 5. Sample processing: Take 100 μL of serum, add 10 μL of internal standard (1 μg / mL trenbolone cyclohexylmethyl carbonate), mix well, add 600 μL of methyl tert-butyl ether, extract on a mixer for 5 minutes, let stand for 1 minute, transfer the upper organic phase to another tube, blow dry under nitrogen, reconstitute with 100 μL of methanol, vortex mix for 1 minute, centrifuge at 14000 rpm for 10 minutes, transfer the supernatant to an injection bottle, inject 5 μL, make a regression curve based on the area ratio of the analyte and the internal standard to the concentration, and calculate the content of the analyte.

[0060] 6. Ultra-high performance liquid chromatography conditions:

[0061] Mobile phase A: 0.1-0.5% formic acid solution; Mobile phase B: methanol (containing 0.1-0.5% formic acid); Chromatographic column model: C18, 2.0×150mm, 3.0μm;

[0062] Gradient elution was used, and the gradient elution process is shown in Table 2:

[0063] Table 2 Gradient elution program

[0064]

[0065] 7. Mass spectrometry conditions:

[0066] Atmospheric pressure chemical ionization (APCI) positive ion mode, multiple reaction monitoring (MRM) was used; the ion source voltage was 4kv (APCI+); the ion transfer tube temperature was 350°C; the nebulizer gas temperature was 350°C, and the sheath gas flow rate was 45L / min. The mass spectrometry ion pairs and parameters are shown in Table 3:

[0067] Table 3 Mass spectrometry ion pairs and parameters

[0068]

[0069]

[0070] 8. The above method was used to measure the normal human serum samples. The results were calculated using the internal standard method. A regression equation was made based on the standard curve: y=ax+b, where y- is the ratio of the peak area of ​​the substance being measured to the peak area of ​​the internal standard, x- is the concentration of the substance being measured, a- is the slope, and b- is the intercept. Each component has a regression equation. The ratio of the peak area of ​​each component of the normal human to the peak area of ​​the internal standard (y) was substituted into the above equation to calculate the concentration of each component (x). The measurement results are shown in Table 4:

[0071] Table 4 Normal human serum sample test results

[0072]

[0073]

[0074] 9. Methodological validation

[0075] a. Linearity: The linear range of the 22 measured components is shown in Table 1, and the linear correlation coefficient (r) is ≥ 0.990.

[0076] b. Recovery rate: The test object was prepared into samples of high, medium and low concentrations with artificial serum, and each concentration was repeated 5 times. The ratio of the result obtained by the above method to the result obtained by the pure standard of the corresponding concentration was multiplied by 100%, which is the recovery rate. The recovery rates of the 23 components (including the internal standard) were all between 93% and 102%.

[0077] c. Repeatability and stability: The test object was prepared into three concentrations of high, medium and low samples with artificial serum. Each concentration was repeated 5 times. The results were measured according to the above method for 3 consecutive days, and the intra-day and inter-day relative standard deviations (RSD) were calculated. The results showed that the intra-day RDS of all 23 components (including internal standard) was ≤6%, and the inter-day RSD was ≤10%.

[0078] The above verifications show that the analytical method of the present invention is accurate.

[0079] The above is a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes according to the technical solutions and concepts of the present invention, which should be covered by the protection scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones, Features: The plurality of fat-soluble vitamins and the plurality of steroid hormones are: testosterone, estrone, progesterone, estradiol, estriol, androstenedione, pregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, 17α-hydroxypregnenolone, 21-hydroxypregnenolone, cortisol, corticosterone, aldosterone, dihydrotestosterone, 11-deoxycortisol, 21-deoxycortisol, vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K; comprising the following steps: Adding internal standard trenbolone cyclohexylmethyl carbonate to the serum sample, mixing well, and then adding an extractant for extraction, wherein the extractant is methyl tert-butyl ether; Liquid chromatography conditions: Mobile phase A: 0.1-0.5% formic acid solution; Mobile phase B: methanol solution containing 0.1-0.5% formic acid; Chromatographic column model: C18; Gradient elution was adopted, and the gradient elution program was as follows: 0-0.5min, the volume percentage of mobile phase B was 45%; 0.5-3min, the volume percentage of mobile phase B gradually decreased to 90%; 3-5min, the volume percentage of mobile phase B was 90%, and from 5-5.1min, the volume percentage of mobile phase B decreased to 45%; 5.1-5.5min, the volume percentage of mobile phase B is 45%; Mass spectrometry conditions: atmospheric pressure chemical ionization source in positive ion mode, using multiple reaction monitoring; ion source voltage was 4 kV, APCI+; ion transfer tube temperature was 340℃-360℃; nebulizer gas temperature was 340℃-360℃, and sheath gas flow rate was 40-50 L / min.

2. The method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones according to claim 1, Features: The ratio of the volume of the extractant to the volume of the sample is (5-10):

1.

3. The method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones according to claim 1 or 2, Features: The extract was blown dry with nitrogen.

4. The method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones according to claim 3, Features: The reconstitution solvent is methanol.

5. The method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones according to claim 1 or 2, Features: The liquid chromatography detection conditions are as follows: Athena C18-WP, 2.1×150 mm, 3 μm; flow rate: 0.4 ml / min; column temperature: 45° C.; mobile phase: A: 0.1% formic acid in water; B: methanol containing 0.1% formic acid.

Images