Method for extracting steroid hormone in serum

Abstract

The invention relates to a method for extracting steroid hormones in serum, and belongs to the technical field of biomedical detection. According to the method, a protein precipitation method is adopted, and methanol, zirconium sulfate heptahydrate and dextran sodium sulfate in a specific proportion are used as protein precipitants, so that protein and phospholipid are effectively settled, the matrix effect is reduced, the detection accuracy is improved, meanwhile, pollution to a chromatographic column and an instrument is reduced, and the service life is prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a method for extracting steroid hormones in serum. Background Art

[0002] Steroid hormones include a large number of small molecules that play a core role in metabolic processes, such as regulatory characteristics, blood pressure and inflammation; their detection and analysis can assist in the diagnosis of neurological disorders with steroid hormone secretion disorders, such as congenital adrenal hyperplasia, polycystic ovary syndrome, adrenal insufficiency and methyphenidyl test, that is, the detection of steroid hormones is of great significance for the diagnosis of a variety of endocrine-related diseases.

[0003] Commonly used detection methods for steroid hormones include traditional radioimmunoassay, direct immunoassay and chromatography-mass spectrometry. However, due to the similarity of the structures of steroid hormones and the existence of isomers, traditional immunoassays have great limitations. Due to its lack of antibody specificity, especially in low concentration ranges, such as in the range of children, and androgens in premenopausal and postmenopausal women, the measured values ​​are high, and only one steroid hormone can be measured at a time. If multiple steroids need to be tested, the sample volume required will be very large. Compared with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has the characteristics of high throughput, high sensitivity and high specificity, and has obvious advantages in the detection of steroid hormones. It only requires a small sample volume to measure multiple steroid hormones at the same time.

[0004] To date, there have been many different LC-MS / MS steroid analysis methods, such as solid phase extraction, liquid-liquid extraction, derivatization and protein precipitation methods, etc., for pre-treatment of serum, and then detection by LC-MS / MS; the pre-treatment steps of liquid-liquid extraction and derivatization are extremely complicated, and the requirements for technicians are relatively high. The solid phase extraction plate of solid phase extraction is expensive, resulting in a relatively high detection cost of this method and a long pre-treatment time, which will correspondingly affect the reporting cycle. Compared with other extraction methods, protein precipitation has the advantages of low cost, simple pre-treatment steps, and short pre-treatment time. However, protein precipitation is to directly take the supernatant for injection after pre-treatment with protein precipitation reagent, which is very easy to damage the chromatographic column and contaminate the mass spectrometer, and soluble impurities such as phospholipids are easy to cause interference, produce ion enhancement or ion suppression, and reduce the accuracy of detection.

[0005] The Chinese patent application with publication number CN111721854A discloses a method for simultaneously detecting 11 steroid hormones in serum. This method is a solid phase extraction method. The pretreatment process mainly uses a releaser A and distilled water to activate the solid phase extraction plate, and then passes the standard curve sample, the quality control sample, and the sample to be tested through the solid phase extraction plate, so that the test substance is adsorbed on the filler of the solid phase extraction plate, and then rinsed with 15% methanol, eluted with acetonitrile, and 5% methanol is added to the eluent and mixed before entering the liquid chromatography-mass spectrometry instrument for analysis. The contents of cortisol, cortisol, 11-deoxycortisol, corticosterone, dehydroepiandrosterone, androstenedione, testosterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, and dehydroepiandrosterone sulfate in the sample are obtained. The pretreatment of the above-mentioned technology uses the principle of solid phase extraction. The disadvantages are: the solid phase extraction plate of the solid phase extraction method is expensive, resulting in a relatively high detection cost of the method, and the pretreatment is time-consuming, which will correspondingly affect the reporting cycle;

[0006] The patent application with application number 2019103397025 applies for a method for simultaneously detecting 13 steroid hormones in serum. This method is a liquid-liquid extraction method. The pretreatment process mainly involves mixing the standard solution with the internal standard solution and then adding methyl tert-butyl ether. After centrifugation by shaking, the supernatant is blown dry with helium and nitrogen, and then re-dissolved with 50% methanol solution and entered into the liquid-mass spectrometer for analysis. The contents of pregnenolone, progesterone, 17a-hydroxypregnenolone, 17a-hydroxyprogesterone, 11a-hydroxyprogesterone, corticosterone, hydrocortisone, cortisone, 11-deoxycortisol, 11-deoxycortisol, 21-deoxycortisol, aldosterone and estrone in the sample are obtained. The above technology uses the principle of liquid-liquid extraction, and the pretreatment requires the upper organic solution to be taken out and blown dry. Disadvantages: The steps are extremely complicated, which is not conducive to the processing of large quantities of samples, and the organic reagents used are extremely volatile, which is more harmful to the experimenters;

[0007] The patent application with application number 202110198064.7 applies for a method for detecting the steroid content in human serum samples. The method is a protein precipitation method. The pretreatment process mainly uses methanol as a solvent, including zinc sulfate and steroid isotope internal standards as protein precipitants. The serum sample to be tested is fully mixed with the protein precipitant containing the internal standard, vortexed and centrifuged to obtain a solution to be tested, and a high performance liquid chromatography-tandem mass spectrometry system is used to detect the solution to be tested; the disadvantages are: using zinc sulfate methanol as a protein precipitator cannot remove phospholipids in the serum, cannot separate interference, and the analyte to be detected is very easy to produce ion enhancement or ion suppression effects in the mass spectrometer, which will reduce the accuracy of the detection, and easily cause contamination to the mass spectrometer and chromatographic column, thereby increasing the high maintenance cost. Summary of the invention

[0008] The purpose of this application is to overcome the above technical problems and provide a method for extracting steroid hormones in serum. The method aims to improve the extraction efficiency, reduce the interference and contamination caused by the protein precipitation method, and improve the accuracy and reliability of the detection cost and detection kit.

[0009] The present application provides a method for extracting steroid hormones in serum, comprising the following steps:

[0010] (1) Sample collection: Collect venous blood to obtain serum samples;

[0011] (2) Protein precipitation: add sodium dextran sulfate solution to the serum sample to be tested, vortex and then add methanol zirconium sulfate solution containing the isotope internal standard of steroid hormones, vortex and shake, shake well and centrifuge, and take the supernatant to obtain the solution to be tested;

[0012] The molecular weight of sodium dextran sulfate in the sodium dextran sulfate solution is 20,000, the solvent is water, and the mass concentration is 50-300 mg / mL; the methanol zirconium sulfate solution includes zirconium sulfate heptahydrate solution and methanol, the solvent of zirconium sulfate heptahydrate is water, and the mass concentration is 50-200 mg / mL; the volume ratio of methanol to zirconium sulfate heptahydrate is any one of 9:1, 8:2, 7:3, and 6:4;

[0013] (3) Use the detection equipment to detect the solution to be tested.

[0014] Through the above technical scheme: the protein precipitation method is used to extract steroid hormones, and the precipitant components are: methanol, zirconium sulfate heptahydrate, sodium dextran sulfate and steroid isotope internal standard. The calibration / quality control / serum samples to be processed are fully mixed with the protein precipitant containing the internal standard, vortexed and centrifuged to obtain the solution to be tested, and the solution to be tested is detected by a high-performance liquid chromatography tandem mass spectrometry system. The sodium dextran sulfate and zirconium ions in this extraction method are combined with methanol to precipitate proteins in serum while also effectively precipitating phospholipids. Compared with the existing protein precipitation method, the matrix effect of the measured steroid hormones is significantly reduced, and the contamination of the chromatographic column and the instrument is also significantly improved. The durability of the chromatographic column is increased from 1500 needles of serum samples to 3000 needles of serum samples, and the mass spectrometry maintenance cycle is also significantly increased.

[0015] Preferably, when the detection device is used for detection, the mobile phase B is 0.05% to 0.1% formic acid methanol, and the mobile phase A is 0.05% to 0.1% formic acid water.

[0016] Preferably, the gradient elution conditions are:

[0017] 0min, mobile phase B was 35%, flow rate was 0.4ml / min;

[0018] 1 min, mobile phase B 35%, flow rate 0.4 ml / min;

[0019] 5min~7.12min, mobile phase B is 65~95%%, flow rate is 0.4~0.5ml / min;

[0020] 8min, mobile phase B was 35%, flow rate was 0.4ml / min.

[0021] Preferably, the instrument used in the liquid chromatography tandem mass spectrometry system is Waters TQS.

[0022] Preferably, the chromatographic column is C18.

[0023] Preferably, the mass spectrometry conditions of the mass spectrometer are: the ion source is an ESI source, and the mode is a positive ion mode.

[0024] Preferably, when the liquid chromatography tandem mass spectrometry system is used for detection, the mobile phase B is 0.1% formic acid methanol, and the mobile phase A is 0.1% formic acid water.

[0025] Preferably, the steroid hormones include androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol, and dehydroepiandrosterone sulfate.

[0026] Preferably, the isotope internal standard of the steroid hormone is androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, dehydroepiandrosterone sulfate-d6, and the solvent is methanol.

[0027] In summary, the present invention has at least the following beneficial effects:

[0028] 1. The combination of sodium dextran sulfate and zirconium ions in this extraction method, combined with methanol, can not only precipitate proteins in serum, but also effectively precipitate phospholipids. Compared with the existing protein precipitation method, the matrix effect of the measured steroid hormones is significantly reduced, and the contamination of chromatographic columns and instruments is also significantly improved. The durability of the chromatographic column is increased from 1500 serum samples to 3000 serum samples, and the mass spectrometer maintenance cycle is also significantly increased.

[0029] 2. In the present invention, a specific liquid chromatography tandem mass spectrometry system and its condition settings are preferably used to achieve efficient separation and detection, and further improve the sensitivity and reliability of detection.

[0030] 3. The method of the present invention is simple, easy to operate, low-cost and causes little harm to experimenters. It is suitable for processing large quantities of samples and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the test result graph of Example 1 of the present application.

[0032] Figure 2 This is a physical observation diagram of the solution to be tested in Examples 1-3 of the present application.

[0033] Reference numerals: Figure 2 Among the 12 solution samples to be tested, from left to right are: 1-3 are the solutions to be tested prepared in Example 1; 4-9 are the solutions to be tested prepared in Example 3; 10-12 are the solutions to be tested prepared in Example 2. DETAILED DESCRIPTION

[0034] The present application is further described below with reference to the accompanying drawings:

[0035] Example 1

[0036] This embodiment discloses a method for extracting steroid hormones in serum, and the specific steps are as follows:

[0037] (1) Sample collection: venous blood was collected using a blood collection tube and centrifuged to obtain a serum sample;

[0038] (2) Preparation of precipitant: Dextran sodium sulfate solution was prepared with a molecular weight of 20,000 Daltons, water as solvent, and a mass concentration of 100 mg / mL; methanol zirconium sulfate solution containing internal standards: the ratio of methanol to zirconium sulfate heptahydrate was 9:1, the concentration of zirconium sulfate heptahydrate was 100 mg / mL, and the solvent was water; the internal standards contained were androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, and dehydroepiandrosterone sulfate-d6;

[0039] (3) Pretreatment: Take 100 μL of serum sample, add 50 μL of 100 mg / mL sodium dextran sulfate solution prepared in step (2), and vortex at 2000 rpm to make it uniform. Then add 100 μL of methanol zirconium sulfate solution containing internal standard, methanol: zirconium sulfate heptahydrate in the methanol zirconium sulfate solution = 9:1 (v / v), and the mass concentration of zirconium sulfate heptahydrate is 100 mg / mL; the internal standard is androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, dehydroepiandrosterone sulfate-d6, and the solvent is methanol, vortex at 2000 rpm for 5 minutes, shake well and then centrifuge for 10 minutes, the centrifugal speed is 14000 rpm, and take the supernatant to obtain the solution to be tested;

[0040] (4) using a liquid chromatography-tandem mass spectrometry system to detect the solution to be tested;

[0041] Liquid chromatography tandem mass spectrometry system detection: Waters TQS instrument was used for detection. Mobile phase B was 0.1% formic acid in methanol, and mobile phase A was 0.1% formic acid in water. The chromatographic column was C18. The mass spectrometry conditions were: the ion source was ESI source, and the mode was positive ion mode. The gradient elution conditions were shown in Table 1.

[0042] Table 1 Gradient elution conditions

[0043]

[0044] Experiments have shown that the durability of the chromatographic column increased from 1500 serum samples to 3000 serum samples, and the mass spectrometer maintenance cycle was also significantly increased. This method has significantly improved the contamination caused by the chromatographic column and instrument.

[0045] Combination Figure 1 It can be seen that in the detection step of the liquid chromatography-tandem mass spectrometry system, multiple steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate are detected simultaneously. By detecting multiple hormones simultaneously, the detection efficiency can be improved and more valuable information can be provided for clinical diagnosis and treatment.

[0046] Experiments have shown that the durability of the chromatographic column increased from 1500 serum samples to 3000 serum samples, and the mass spectrometer maintenance cycle was also significantly increased. This method has significantly improved the contamination caused by the chromatographic column and instrument.

[0047] Combination Figure 1 It can be seen that in the detection step of the liquid chromatography-tandem mass spectrometry system, multiple steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate are detected simultaneously. By detecting multiple hormones simultaneously, the detection efficiency can be improved and more valuable information can be provided for clinical diagnosis and treatment.

[0048] Example 2

[0049] (1) Sample collection: venous blood was collected using a blood collection tube and centrifuged to obtain a serum sample;

[0050] (2) Preparation of precipitant: The internal standard dextran sodium sulfate solution was prepared with a molecular weight of 20,000 Daltons, a solvent of water, and a mass concentration of 100 mg / mL; the internal standards contained were androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, and dehydroepiandrosterone sulfate-d6;

[0051] (3) Pretreatment: Take 100 μL of serum sample, add 150 μL of 100 mg / mL sodium dextran sulfate solution prepared in step (2), vortex at 2000 rpm for 5 min; shake well and centrifuge, take the supernatant to obtain the test solution;

[0052] (4) The solution to be tested is tested using a liquid chromatography-tandem mass spectrometry system; the testing conditions are the same as those of step (4) of Example 1.

[0053] Example 3

[0054] (1) Sample collection: venous blood was collected using a blood collection tube and centrifuged to obtain a serum sample;

[0055] (2) Preparation of precipitant: Prepare a methanol zirconium sulfate solution containing internal standards: the ratio of methanol to zirconium sulfate heptahydrate is 9:1 (v / v), the concentration of zirconium sulfate heptahydrate is 100 mg / mL, and the solvent is water; the internal standards contained are androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, and dehydroepiandrosterone sulfate-d6;

[0056] (3) Pretreatment: Take 100 μL of serum sample, add 150 μL of the methanol zirconium sulfate solution containing the internal standard prepared in step (2), vortex at 2000 rpm for 5 min, shake well and centrifuge, and take the supernatant to obtain the solution to be tested;

[0057] (4) using a liquid chromatography-tandem mass spectrometry system to detect the solution to be tested;

[0058] Take the solution to be tested in Example 1-3, place it at room temperature for 10 minutes and then observe it. Figure 2 When the serum samples were pre-treated by the methods of Example 1 and Example 3, the solution to be tested had obvious precipitation and stratification. When the method of Example 2 was used, no obvious precipitation and stratification was produced in the solution to be tested.

[0059] The extraction methods of Example 1 and Example 3 were evaluated for matrix effect in the following steps:

[0060] Select three surrogate matrix samples of low, medium and high concentrations containing androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate, six clinical samples of human serum from different sources, and a 1:1 mixture of the two, and treat them with the methods of Example 1 and Example 3 respectively, and treat 3 samples of each type in parallel for sample analysis. Investigate the deviation of the response value of the mixed sample from the mean value of the response value of the clinical sample and the pure solution sample. Obtain Table 1 Table 1 Example 1 Extraction method matrix effect result table and Table 2 Example 3 Extraction method matrix effect result table.

[0061] Table 1 Example 1 Extraction method matrix effect results table

[0062]

[0063]

[0064] Table 2 Matrix effect results of Example 3 extraction method

[0065]

[0066] As can be seen from Table 1, the extraction method of Example 1 shows that in the six human sera from different sources, the matrix effects of the low, medium and high concentrations of androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate are all less than 15%; as can be seen from Table 2, the extraction method of Example 3 shows that dehydroepiandrosterone sulfate has an obvious matrix enhancement effect, and testosterone and progesterone also have a matrix enhancement effect in some sera; analysis shows that the extraction method of Example 1 has a better impurity removal effect.

[0067] After testing, the mass concentration of sodium dextran sulfate in Example 1 is between 50 and 300 mg / mL, and various steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate can be detected. When the mass concentration of sodium dextran sulfate is 100 mg / mL, the effect is best, and the detection efficiency and accuracy are high.

[0068] After testing, the mass concentration of the zirconium sulfate heptahydrate aqueous solution in Example 1 is between 50 and 200 mg / mL and 50 and 300 mg / mL, and various steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate can be detected. When the mass concentration of the zirconium sulfate heptahydrate aqueous solution is 100 mg / mL, the effect is best, and the detection efficiency and accuracy are high.

[0069] When the volume ratio of methanol to zirconium sulfate heptahydrate in Example 1 is 9:1, 8:2, 7:3, and 6:4, various steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol, and dehydroepiandrosterone sulfate can be detected. When the volume ratio of methanol to zirconium sulfate heptahydrate is 9:1, the effect is best, and the detection efficiency and accuracy are high.

[0070] When the concentration of the flowable A formic acid water in Example 1 is in the range of 0.05% to 0.1%, various steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate can be detected. When the concentration of the flowable A formic acid water is in the range of 0.1%, the effect is the best, and the detection efficiency and accuracy are high.

[0071] When the concentration of the formic acid water of the flowability B in Example 1 is in the range of 0.05% to 0.1%, various steroid hormones such as androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol and dehydroepiandrosterone sulfate can be detected. When the concentration of the formic acid water of the flowability B is in the range of 0.1%, the effect is the best, and the detection efficiency and accuracy are high.

[0072] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for extracting steroid hormones from serum, characterized in that: The steps include: (1) Sample collection: Collect venous blood to obtain serum samples; (2) Precipitating agent preparation: preparing a sodium dextran sulfate solution and a methanol zirconium sulfate solution; wherein the molecular weight of the sodium dextran sulfate in the sodium dextran sulfate solution is 20,000 Daltons, the solvent is water, and the mass concentration is 50 to 300 mg / mL; the methanol zirconium sulfate solution includes a zirconium sulfate heptahydrate solution and methanol, the solvent of the zirconium sulfate heptahydrate is water, and the mass concentration is 50 to 200 mg / mL; the volume ratio of methanol to zirconium sulfate heptahydrate is any one of 9:1, 8:2, 7:3, and 6:4; (3) Pretreatment: add sodium dextran sulfate solution to the serum sample to be tested, vortex and then add methanol zirconium sulfate solution containing the isotope internal standard of steroid hormones, vortex and shake, shake well and centrifuge, and take the supernatant to obtain the solution to be tested; (4) Use the detection equipment to detect the solution to be tested.

2. The extraction method according to claim 1, characterized in that When the detection equipment is used for detection, the mobile phase B is 0.05% to 0.1% formic acid methanol, and the mobile phase A is 0.05% to 0.1% formic acid water.

3. The extraction method according to claim 2, characterized in that The gradient elution conditions were: 0min, mobile phase B was 35%, flow rate was 0.4ml / min; 1 min, mobile phase B 35%, flow rate 0.4 ml / min; 5min~7.12min, mobile phase B is 65~95%%, flow rate is 0.4~0.5ml / min; 7.2~8min, mobile phase B is 35%, flow rate is 0.4ml / min.

4. The extraction method according to claim 3, characterized in that The instrument used in the liquid chromatography tandem mass spectrometry system is Waters TQS.

5. The extraction method according to claim 3, characterized in that The chromatographic column used was C18.

6. The extraction method according to claim 1, characterized in that The mass spectrometry conditions used were as follows: the ion source was ESI source and the mode was positive ion mode.

7. The extraction method according to claim 1, characterized in that The steroid hormones include androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisol, and dehydroepiandrosterone sulfate.

8. The extraction method according to claim 1, characterized in that The isotope internal standards of the steroid hormones are androstenedione-13C3, testosterone-13C3, progesterone-d9, 17-hydroxyprogesterone-d8, cortisol-d4, and dehydroepiandrosterone sulfate-d6, and the solvent is methanol.

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