Rapid detection method for hormones related to polycystic ovary

Abstract

The invention discloses a rapid detection method for hormones related to polycystic ovary, and particularly relates to the technical field of hormone detection. The method comprises the following steps: drawing a standard curve by adopting a stable isotope internal standard quantification method and taking the concentration of a standard substance of polycystic ovary related hormones as an X axis and a peak area ratio corresponding to an internal standard in a calibrator as a vertical coordinate (y); processing the sample, and calculating and obtaining the concentration of the hormone in the serum sample by using a standard curve; the hormones related to the polycystic ovary comprise corticosterone, cortisone, cortisol, 18-hydroxycortisol, androstenedione, 11-hydroxyandrostenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate. The method utilizes high performance liquid chromatography-tandem mass spectrometry for detection, and has the advantages of high detection speed and high sensitivity. An accurate detection result can be obtained in a short time, and timely support is provided for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of hormone detection, and in particular to a rapid detection method for hormones associated with polycystic ovary. Background Art

[0002] Hormones play crucial roles at the endocrine, paracrine, and endocrine regulatory levels of the body. For example, corticosteroids (e.g., glucocorticoids and mineralocorticoids) are involved in a variety of biological processes, including stress and immune responses as well as regulation of inflammation and carbohydrate metabolism; sex steroids (e.g., progesterone, androgens, and estrogens) mediate the development of reproductive function and sexual characteristics, among other functions; and melatonin is involved in synchronizing circadian rhythms, including sleep-wake time, blood pressure regulation, and seasonal reproduction.

[0003] Polycystic ovary syndrome (PCOS), also known as Stein-Leventhal syndrome, is the most common endocrine and metabolic disease in women of childbearing age, and its causes may be related to genetic and environmental factors. PCOS is a collection of a diverse, multi-system chronic endocrine disorder, such as irregular menstruation, hirsutism, obesity, hyperlipidemia, various manifestations of hyperandrogenism, cystic follicles, insulin resistance and infertility, etc. Most patients only have prominent manifestations of a few of these, and clinical heterogeneity is the main reason why there is no unified diagnostic standard.

[0004] At present, chemiluminescence or immunoassay is mainly used clinically to detect steroid hormones to determine hyperandrogenemia. However, due to the low content and similar structure of hormones such as steroids related to polycystic ovary syndrome in serum, conventional chemiluminescence or immunoassay methods will cause false positive or false negative results of androgen detection due to factors such as low sensitivity, poor specificity and cross-reaction of the detection system, which will affect the doctor's judgment of the condition. Chinese patent CN114609297A discloses a method for simultaneously detecting 5 steroid hormones based on high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Although this method can separate and quantify A4, T, DHT, 17-OHP and DHEAS to a certain extent, the upper limit of quantification of DHEAS is low, and it is impossible to achieve quantitative detection of high concentration DHEAS in the sample, and further dilution is required for detection; and the lower limits of quantification of T, DHT and 17-OHP are high, and it is impossible to achieve quantitative detection of low concentrations of the substance in the sample, so the overall clinical detection needs cannot be met.

[0005] Chinese patent CN115219617A, a multi-hormone synchronous detection method for PCOS, includes the following steps: S100, establish a standard curve of 7 hormones; S200, add an internal standard to the biological sample to be detected, and mix thoroughly to form a first mixture; S300, add a protein precipitant to form a second mixture, and obtain a supernatant; S400, add a diluent to form a third mixture; S500, perform solid phase extraction on the third mixture, collect a solution containing a target component; S600, perform high performance liquid chromatography tandem mass spectrometry detection on the target component, collect signals, and calculate concentrations. However, this method still cannot achieve quantitative detection of low concentrations of the substance in the sample, so it cannot meet clinical detection needs as a whole. Summary of the invention

[0006] To this end, the present invention provides a rapid detection method for hormones related to polycystic ovary to solve the above-mentioned problems.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] According to the present invention, a rapid detection method for hormones related to polycystic ovary is provided, the method comprising:

[0009] Step 1, using the stable isotope internal standard quantitative method, the concentration of the standard substance of the hormone related to polycystic ovary is used as the X-axis, and the peak area ratio of the corresponding internal standard in the calibrator is used as the ordinate (y) to draw a standard curve;

[0010] Step 2: Sample processing

[0011] Serum samples were added with internal standard substances of hormones related to polycystic ovary, precipitant, and purified water, respectively, and centrifuged after oscillation and mixing. The supernatant was taken and dried in a solid phase extraction plate, and then eluted with eluent 1 and eluent 2, respectively, filtered and dried with a positive pressure device. Finally, KS-elutant 1 was taken and filtered with a positive pressure device in a solid phase extraction plate, and the eluent was received by a collection plate added with purified water, and detected by high performance liquid chromatography tandem mass spectrometry;

[0012] Step 3, using the standard curve, calculating and obtaining the concentration of the hormone in the serum sample;

[0013] The hormones associated with polycystic ovary include corticosterone, cortisone, cortisol, 18-hydroxycortisol, androstenedione, 11-hydroxyandrostenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

[0014] Furthermore, in step 2, the HPLC detection conditions are as follows: mobile phase A is 0.02-0.04% ammonium fluoride aqueous solution, mobile phase B is pure methanol, and gradient elution is performed. As an example, preferably mobile phase A is 0.03% ammonium fluoride aqueous solution.

[0015] Furthermore, the conditions of the gradient elution are as follows: when the flow rate of the mobile phase is 0.2-0.4 mL / min, the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5) during 0.50-1 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5) during 1-4 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, the volume ratio of the mobile phase A to the mobile phase B is (4-5):(5-6); at 6-7 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (2-3):(7-8); at 6.55-7.2 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (5-15):(85-95);

[0016] At 7.25-9min, the flow rate of the mobile phase is 0.2-0.4mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5).

[0017] As an example, preferably, during 0-0.50 min, the flow rate of the mobile phase is 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; during 0.50 min, the flow rate of the mobile phase is 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; during 4 min, the flow rate of the mobile phase is 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is 45:55;

[0018] At 6.5 min, the flow rate of the mobile phase was 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B was 25:75; at 6.55-7.2 min, the flow rate of the mobile phase was 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B was 10:90; at 7.25-9 min, the flow rate of the mobile phase was 0.3 mL / min, and the volume ratio of the mobile phase A to the mobile phase B was 60:40.

[0019] Furthermore, the mass spectrometry conditions are: nebulizing gas (Gas1) is 50 psi, auxiliary gas (Gas2) is 65 psi, curtain gas (CUR) is 25 psi, collision gas (CAD) is 9 psi, and ion source temperature is 450°C.

[0020] As an example, the detection ion pair of corticosterone is 347.1 / 91.3; the detection ion pair of cortisone is 361.3 / 163.1; the detection ion pair of cortisol is 363.2 / 121.0; the detection ion pair of 18-hydroxycortisol is 363.1 / 269.2; the detection ion pair of androstenedione is 287.4 / 97.0; the detection ion pair of 11-hydroxyandrostenedione is 303.1 / 267.2; the detection ion pair of dehydroepiandrosterone is 271.1 / 253.0; and the detection ion pair of dehydroepiandrosterone sulfate is 367.1 / 80.0.

[0021] Furthermore, in the step 2, before using the solid phase extraction plate, the activation solution and purified water are respectively transferred into the solid phase extraction plate, filtered and dried.

[0022] Furthermore, in the step 2, the activation solution is methanol.

[0023] Furthermore, in step 2, the eluents 1 and 2 are mixtures of methanol and water in different proportions.

[0024] Furthermore, in step 2, eluent 1 is methanol: water = 0.5-1.5; eluent 2 is methanol: water = 2-3. As an example, preferably eluent 1 is methanol: water = 1:1; eluent 2 is methanol: water = 7:3.

[0025] Furthermore, in step 2, the precipitant is methanol: water = 0.5-1.5. As an example, the preferred precipitant is methanol: water = 1.

[0026] Furthermore, in step 2, the conditions for oscillation and mixing are 2000-3000 r / min for 0.5-1.5 min. As an example, preferably 2500 r / min for 10 min.

[0027] The present invention has the following advantages:

[0028] The present invention adopts the stable isotope internal standard quantitative method, and draws the standard curve with the concentration of the hormone standard as the X-axis and the peak area ratio of the corresponding internal standard in the calibrator as the ordinate, which can effectively reduce the error in the detection process. The stable isotope internal standard can correct the loss and variation in the sample processing process, ensuring the accuracy and reliability of the test results.

[0029] The present invention undergoes strict sample processing steps, including adding internal standard, precipitant and purified water, shaking and mixing, centrifugation, drying and multiple washing and filtering operations. These steps can remove impurities in the serum sample, improve the specificity of the test, and make the test results more accurately reflect the content of the target hormone.

[0030] The method of the present invention can simultaneously detect a variety of hormones related to polycystic ovary, including corticosterone, cortisone, cortisol, 18-hydroxycortisol, androstenedione, 11-hydroxyandrostenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate. This is crucial for a comprehensive understanding of the endocrine status of polycystic ovary patients. Different hormones may play different roles in the pathogenesis of polycystic ovary. By detecting these hormones simultaneously, more comprehensive diagnostic information can be provided to doctors, which helps to formulate more accurate treatment plans.

[0031] Although the sample processing process of the present invention has many steps, the operation is relatively simple and does not require complex instruments and professional technicians. Through standardized operating procedures, the pre-treatment of samples can be completed quickly and accurately. In addition, the present invention uses high performance liquid chromatography tandem mass spectrometry for detection, which has the advantages of fast detection speed and high sensitivity. Accurate test results can be obtained in a relatively short time, providing timely support for clinical diagnosis and treatment.

[0032] The method of the present invention has been rigorously experimentally verified and optimized, and has high reliability. Each step of drawing the standard curve and sample processing has been carefully designed and verified to ensure the stability and repeatability of the test results. The present invention can effectively avoid the contamination and loss of the sample through operations such as filtering and drying by a positive pressure device, thereby ensuring the reliability of the test results. At the same time, adding purified water to the collection plate to receive the eluent also helps to improve the accuracy of the test.

[0033] The present invention detects 8 hormones related to polycystic ovary, which has important guiding significance for the diagnosis and treatment of polycystic ovary patients. By accurately detecting the levels of multiple hormones in the patient's serum, it can help doctors determine the severity of the disease, evaluate the treatment effect, and formulate personalized treatment plans. It can be used to monitor the progression of the disease and the response to treatment. Regularly testing the patient's hormone level can timely detect changes in the disease, adjust the treatment plan, and improve the success rate of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0035] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0036] Figure 1 An experimental spectrum (peak diagram) of 8 hormone analytes provided in Example 1 of the present invention;

[0037] Figure 2 Serum sample chromatograms of 8 analytes provided in Example 8 of the present invention and their respective retention time graphs;

[0038] Among them, A-DHEA; B-A4; C-11-OH-A4; D-Corticosterone; E-Cortisone; F-Cortisol; G-18-0H-corlisol; H-DHEAS. DETAILED DESCRIPTION

[0039] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Hormones related to polycystic ovary are shown in Table 1 .

[0041] Table 1

[0042] Chinese name English name abbreviation DHEA Dehydroepiandrosterone DHEA Androstenedione Androstenedione A4 11-Hydroxyandrostenedione 11-Hydroxyandrostenedione 11-OH-A4 Corticosterone Corticosterone Corticosterone Cortisone Cortisone Cortisone Cortisol Cortisol Cortisol 18-Hydroxycortisol 18-Hydroxycortisol 18-OH-cortisol Dehydroepiandrosterone Sulfate Dehydroepiandrosterone sulfate DHEAS

[0043] 1. Materials

[0044] Source of experimental samples: Nanning Second People's Hospital;

[0045] 2. Instruments and main consumables

[0046] Hangzhou Kailai HPLC tandem mass spectrometry detection system CalQuant-S; centrifuge: Beijing Baiyang medical centrifuge BY-R16; vortexer: Hangzhou Ruicheng multi-tube vortex mixer MS200; chromatographic grade methanol: Fisher (A452-4);

[0047] 3. Methods

[0048] 1) Preparation of relevant solutions

[0049] Standards: Accurately weigh 11.2032 mg of ketone, 12.3257 mg of cortisone, 10.4267 mg of cortisol, 17.789 mg of 18-hydroxycortisol, 15.4578 mg of androstenedione, 13.4257 mg of 11-hydroxyandrostenedione, 13.3573 mg of dehydroepiandrosterone and 15.5257 mg of dehydroepiandrosterone sulfate, respectively, and add them into a 100 ml volumetric flask at the same time, fully dissolve, shake well, and separate. The target concentrations were 0.112032 mg / ml, 0.123257 mg / ml, 0.104267 mg / ml, 0.17789 mg / ml, 0.154578 mg / ml, 0.134257 mg / ml, 0.133573 mg / ml and 0.155257 mg / ml respectively, which were mixed and then diluted with methanol to form mixed standard solutions of different concentrations and stored at -80°C for use.

[0050] Preparation of internal standard solution: Prepare the internal standard solution in the same way as above.

[0051] Blank serum: PBS was used as blank serum, manufacturer: Thermo Fisher Scientific (China) Co., Ltd., batch number: 20240110.

[0052] Sample to be tested: Take the serum sample to be tested, add the internal standard solution to obtain the solution to be tested;

[0053] Standard test solution: Add internal standard solution and mixed standard solutions of different concentrations into blank serum to obtain standard test solutions of different concentrations.

[0054] Mobile phase A: 0.03% ammonium fluoride aqueous solution;

[0055] Activation solution: methanol;

[0056] Eluent 1 is methanol: water = 1:1;

[0057] Eluent 2 was methanol: water = 7:3;

[0058] The precipitant is methanol: water = 1.

[0059] 2) Extraction of hormones to be tested

[0060] Samples to be tested: Take 200μL of the sample to be tested, add 20μL of the internal standard, oscillate at 2500r / min for 10min. Add 200μL of precipitant (1:1 methanol: water), oscillate at 2500r / min for 1min; then add 300μL of purified water, oscillate at 2500r / min for 1min. Place the sample in a centrifuge and centrifuge at 12000rpm for 25min. Then, take 650μL of activation solution (methanol) and purified water in the solid phase extraction plate, filter and blow dry with a positive pressure device, then take 500μL of supernatant in the solid phase extraction plate and blow dry in the same way; then take 500μL of eluent 1 (1:1 methanol: water) and eluent 2 (7:3 methanol: water) in turn for elution operation and filter and blow dry with a positive pressure device. Finally, accurately transfer 60 μL of KS-elution solution 1 (1:1 methanol: water) to the solid phase extraction plate and filter it through a positive pressure device. Receive the eluate with a 96-well collection plate to which 70 μL of purified water has been added. Apply a sticky sealing film and mix for 2 minutes.

[0061] The method for extracting hormones from the standard test solution is the same as above.

[0062] 3) LC-MS analysis

[0063] Chromatographic conditions: Column: Waters ACQUITY BEH C8 1.7μm 2.1x 100mm Column; mobile phase A is 0.03% ammonium fluoride aqueous solution; mobile phase B is pure methanol; injection volume is 10μL.

[0064] The gradient elution is shown in Table 2.

[0065] Table 2

[0066] time A(％) B(％) Flow rate (mL min-1) 0 60 40 0.3 0.5 60 40 0.3 4 45 55 0.3 6.5 25 75 0.3 6.55 10 90 0.3 7.2 10 90 0.3 7.25 60 40 0.3 8.8 60 40 0.3 9 60 40 0.3

[0067] The mass spectrometry conditions are as follows: the mass spectrometry detector operating parameters are set as follows: nebulizer gas (Gas1) is 50 psi, auxiliary gas (Gas2) is 65 psi, curtain gas (CUR) is 25 psi, collision gas (CAD) is 9 psi, and ion source temperature is 450° C. The mass spectrometry acquisition parameters of the analytes are shown in Table 3:

[0068] Table 3

[0069]

[0070] The following methodological validation was carried out in terms of precision, limit of quantification, linearity and carryover effect.

[0071] Example 1

[0072] This embodiment provides a selective verification method:

[0073] Accurately weigh 11.2032 mg of ketone, 12.3257 mg of cortisone, 10.4267 mg of cortisol, 17.789 mg of 18-hydroxycortisol, 15.4578 mg of androstenedione, 13.4257 mg of 11-hydroxyandrostenedione, 13.3573 mg of dehydroepiandrosterone and 15.5257 mg of dehydroepiandrosterone sulfate, respectively, and add them into a 100 ml volumetric flask at the same time, fully dissolve, shake well, and obtain the target concentration of 0.11 2032mg / ml, 0.123257mg / ml, 0.104267mg / ml, 0.17789mg / ml, 0.154578mg / ml, 0.134257mg / ml, 0.133573mg / ml and 0.155257mg / ml, standard solutions, were mixed with blank serum, extracted by the test hormone extraction method, and compared with the hormone standard alone, e.g. Figure 2 No interference shown.

[0074] Example 2

[0075] This example provides an accuracy verification experiment:

[0076] Use high-value and low-value accuracy standards to verify accuracy, compare the test results with the known correct results, repeat the test 3 times, and measure the quality control products of the kit at the same time. When the quality control results are under control, record the test value of the sample. Two sets of test data are obtained for each indicator, and the relative deviation is calculated. The relative deviation of the low-value accuracy standard is ≤20.0%, and the relative deviation of the high-value accuracy standard is ≤15.0%, then the accuracy verification is considered to have passed. The results are shown in Table 4.

[0077] Table 4

[0078]

[0079]

[0080] The relative deviation of the low-value accuracy standard for each test indicator is ≤20.0%, and the relative deviation of the high-value accuracy standard is ≤15.0%, which meets the acceptance criteria and passes the verification.

[0081] Example 3

[0082] This example provides intra-batch precision verification:

[0083] Use high-value and low-value precision standards to verify the intra-batch precision. Repeat the test 10 times in the same batch of experiments, and measure the quality control products of the kit at the same time. When the quality control results are under control, record the test values ​​of the samples. A total of 2 sets of test data are obtained for each test indicator, and the coefficient of variation (CV) is calculated. If the CV of the low-value precision standard is ≤20.0% and the CV of the high-value precision standard is ≤15.0%, the intra-batch precision verification is considered to have passed. The results are shown in 5.

[0084] Table 5

[0085]

[0086]

[0087]

[0088] The CV of the low-precision standard products of the eight hormone indicators of the present invention is ≤20.0%, and the CV of the high-precision standard products is ≤15.0%, and the verification is passed.

[0089] Example 4

[0090] This example provides quantitative detection limit verification:

[0091] The quantitative limit (sensitivity) was verified using quantitative limit standards, and the test results were compared with the known correct results. The test was repeated 5 times, and the quality control product of the kit was determined at the same time. When the quality control results were under control, the test values ​​of the samples were recorded. A set of test data was obtained for each indicator, and the average relative deviation and CV were calculated. If the average relative deviation was ≤20.0% and the CV was ≤20.0%, the quantitative detection lower limit was considered to have passed the verification. The results are shown in Table 6.

[0092] Table 6

[0093]

[0094]

[0095] The average relative deviation of the eight hormone indicators of the present invention is ≤20.0% and the CV is ≤20.0%, and the sensitivity verification is passed.

[0096] Example 5

[0097] This example verifies the linear range:

[0098] Linear standards 1-6 produced by a third party were used to verify the linear range, and the test results were compared with the known correct results. The test was repeated 3 times, and the quality control product of the kit was determined at the same time. When the quality control results were under control, the test values ​​of the samples were recorded. Six sets of test data were obtained for each test indicator, and the correlation coefficient, relative deviation and absolute deviation were calculated. The results are shown in Tables 7 and 8.

[0099] Table 7

[0100]

[0101]

[0102]

[0103] Table 8

[0104] Detection object Regression equation Correlation coefficient Linear range (ng / mL) DHEA Y=0.73008X+0.00597 0.99705 0.3-90 Androstenedione Y=1.47129X+0.05634 0.99775 0.1-30 11-Hydroxyandrostenedione Y=0.19967X-0.00123 0.99751 0.05-15 Corticosterone Y=0.52937X+0.00449 0.99572 0.1-30 Cortisone Y=1.25819X+0.20868 0.99934 0.55-165 Cortisol Y=0.43737X+0.14842 0.99865 1-300 18-Hydroxycortisol Y=0.14647X-0.00228 0.99784 0.05-30 Dehydroepiandrosterone Sulfate Y=0.00316X+0.00044 0.99951 25-7500

[0105] Results Figure 1 As shown, DHEA: sample peak 271.1 / 253.0, internal standard peak 294.2 / 258.2; A4: sample peak 287.4 / 97.0, internal standard peak 294.2 / 100.2; 11-OH-A4: sample peak 303.1 / 267.2, internal standard peak 310.2 / 274.2; Corticosterone: sample peak 347.1 / 91.3, internal standard peak 351.3 / 273.2; Co Cortisone: sample peak 361.3 / 163.1, internal standard peak 369.2 / 125.2; Cortisol: sample peak 363.2 / 121.0, internal standard peak 367.1 / 313.3; 18-0H-corlisol: sample peak 379.1 / 267.1, internal standard peak 383.2 / 271.2; DHEAS: sample peak 367.1 / 80.0, internal standard peak 373.2 / 80.0.

[0106] Example 7

[0107] This embodiment provides verification of the carryover effect:

[0108] Carryover effect verification was performed using the highest concentration point C6 of the standard curve of the kit. After injection, a blank sample was injected. The cycle was repeated three times, and the sample peak area and internal standard peak area in the blank sample were recorded. The residual in the blank sample after the highest point of the calibration sample injection should not exceed 20% of the lower limit of quantitation and should not exceed 5% of the internal standard, which is considered to be verified. The results are shown in Table 9.

[0109] Table 9

[0110]

[0111]

[0112] The residues in the blank samples after the highest point injection of each index calibrant of the present invention do not exceed 20% of the quantitative lower limit, and do not exceed 5% of the internal standard, which meets the standard and passes the verification.

[0113] Example 8

[0114] The standard test solution was methodologically validated in terms of accuracy, lower limit of quantification, linearity and carryover effect. The results are shown in Table 10.

[0115] Table 10

[0116]

[0117]

[0118] The standard of the accuracy of the standard test solution of the present invention is less than 15%; the standard of the quantitative lower limit is to simultaneously meet the average relative deviation ≤ 20.0% and CV ≤ 20.0%; when the blank sample is repeatedly injected at the highest point of the calibrant, it is considered that there is no obvious carryover contamination if the residual amount does not exceed 20% of the quantitative lower limit; when the correlation coefficient (R) is ≥ 0.99 and the linear relative deviation is within ± 15.0%, the calibration curve is considered to be linear.

[0119] The serum sample chromatograms of 8 analytes and their respective retention times are shown in Figure 2. Figure 2 As shown. The horizontal axis is time (min), the vertical axis is intensity (cps), and the retention time of the analytes is listed in the order of elution. (1) 18-OH-cortisol, 3.28min; (2) Cortisone, 4.22min; (3) Cortisol, 4.62min; (4) DHEAS, 4.68min; (5) 11-OH-A4, 5.10min; (6) 11-OH-A4, 5.10min; (7) Corticosterone, 5.62min; (8) A4, 6.34min; (9) DHEA, 6.82min. It can be seen that the eight hormones do not interfere with each other.

[0120] Example 9

[0121] The present invention is the first to establish a LC-MS / MS quantitative method for the normal value range of hormone rapid detection methods related to polycystic ovary. According to the peak graph, the spike recovery rates of the eight indicators are all within the range of 85% to 115%; the coefficient of variation within the batch is no more than 15.0%. The specific normal value range is determined as shown in Table 11:

[0122] Table 11

[0123]

[0124] Experimental Example 1

[0125] Using the rapid detection method for hormones associated with polycystic ovary of the present invention, serum samples of 450 patients with polycystic ovary (patient source: Nanning Second People's Hospital) were monitored and analyzed. According to age classification, 150 people were aged 18-25, 150 people were aged 25-35, and 150 people were aged 35-45. All polycystic ovary patients fell within the range given in Example 9, and the detection accuracy reached 100%. Sera of 1,000 non-polycystic ovary volunteers were taken, and at least one indicator was not within the range of Example 9, indicating that the rapid detection index of hormones associated with polycystic ovary established in Example 9 of the present invention has high sensitivity and high accuracy.

[0126] Serum samples from 19 of the 450 women of childbearing age were blindly collected, and the test data are shown in Table 12.

[0127] Table 12

[0128]

[0129]

[0130] Comparative Example 1

[0131] The method for simultaneously detecting 8 steroid hormones by ultra-high performance liquid chromatography-tandem mass spectrometry provided in this comparative example has similar steps to those in Example 4, and both the chromatographic conditions and mass spectrometry parameters have been optimized. The only difference is the elution gradient. The elution conditions are shown in Table 13:

[0132] Table 13

[0133] time A(％) B(％) Flow rate (mL min-1) 0 55 45 0.3 0.5 55 45 0.3 4 45 55 0.3 6.5 25 75 0.3 6.55 15 85 0.3 7.2 15 85 0.3 7.25 75 25 0.3 8.8 55 45 0.3 9 55 45 0.3

[0134] Compared with Example 4, the sensitivity of the eight steroid hormones in Comparative Example 1 decreased. Specifically, the sensitivity (based on the minimum detection limit) decreased by 10%, as shown in Table 14.

[0135] Table 14

[0136]

[0137] Comparative Example 2

[0138] In the extraction method of the hormone to be tested, the recovery rate is different for different parameters, as follows:

[0139] The precipitant adopts methanol: water = 0.5, and the recovery rate is 85%; methanol: water = 1.5, and the recovery rate is 85%; preferably, the methanol: water of the present invention = 1, and the recovery rate is 90%.

[0140] The eluent 1 uses methanol: water = 0.5, with a recovery rate of 85%; methanol: water = 1.5, with a recovery rate of 85%; preferably, the methanol: water of the present invention = 1, with a recovery rate of 90%.

[0141] The eluent 2 uses methanol: water = 2, with a recovery rate of 85%; methanol: water = 3, with a recovery rate of 85%; preferably, the methanol: water = 7:3 of the present invention, with a recovery rate of 90%.

[0142] The activation solution is ethanol, and the recovery rate is 85%; preferably, the activation solution of the present invention is methanol, and the recovery rate is 90%.

[0143] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A rapid test method for hormones related to polycystic ovary, characterized in that: The method comprises: Step 1, using the stable isotope internal standard quantitative method, the concentration of the standard substance of the polycystic ovary-related hormone is used as the X-axis, and the peak area ratio of the corresponding internal standard in the calibrator is used as the ordinate (y) to draw a standard curve; Step 2: Sample processing Serum samples were added with internal standard substances of hormones related to polycystic ovary, precipitant, and purified water, respectively, and centrifuged after oscillation and mixing. The supernatant was taken and dried in a solid phase extraction plate, and then eluted with eluent 1 and eluent 2, respectively, filtered and dried with a positive pressure device. Finally, KS-elutant 1 was taken and filtered with a positive pressure device in a solid phase extraction plate, and the eluent was received by a collection plate added with purified water, and detected by high performance liquid chromatography tandem mass spectrometry; Step 3, using the standard curve, calculating and obtaining the concentration of the hormone in the serum sample; The hormones associated with polycystic ovary include corticosterone, cortisone, cortisol, 18-hydroxycortisol, androstenedione, 11-hydroxyandrostenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

2. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: In the step 2, the HPLC detection conditions are as follows: mobile phase A is 0.02-0.04% ammonium fluoride aqueous solution, mobile phase B is pure methanol, and gradient elution is performed.

3. A rapid test method for hormones related to polycystic ovary according to claim 2, characterized in that: The conditions of the gradient elution are as follows: when the flow rate of the mobile phase is 0.2-0.4 mL / min during 0-0.50 min, the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5); when the flow rate of the mobile phase is 0.2-0.4 mL / min during 0.50-1 min, the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5); when the flow rate of the mobile phase is 1-4 min, the volume ratio of the mobile phase A to the mobile phase B is (4-5):(5- 6); at 6-7 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (2-3):(7-8); at 6.55-7.2 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (5-15):(85-95); at 7.25-9 min, the flow rate of the mobile phase is 0.2-0.4 mL / min, and the volume ratio of the mobile phase A to the mobile phase B is (5-7):(3-5).

4. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: The mass spectrometry conditions are as follows: nebulizing gas (Gas1) is 50 psi, auxiliary gas (Gas2) is 65 psi, curtain gas (CUR) is 25 psi, collision gas (CAD) is 9 psi, and ion source temperature is 450°C.

5. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: In the step 2, before using the solid phase extraction plate, the activation solution and purified water are respectively transferred into the solid phase extraction plate for filtration and drying.

6. A rapid test method for hormones related to polycystic ovary according to claim 5, characterized in that: In the step 2, the activation solution is methanol.

7. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: In the step 2, the eluents 1 and 2 are respectively mixtures of methanol and water in different proportions.

8. A rapid test method for hormones related to polycystic ovary according to claim 7, characterized in that: In the step 2, the eluent 1 is methanol: water = 0.5-1.5; the eluent 2 is methanol: water = 2-3.

9. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: In the step 2, the precipitant is methanol: water = 0.5-1.

5.

10. A rapid test method for hormones related to polycystic ovary according to claim 1, characterized in that: In the step 2, the conditions for oscillation mixing are 2000-3000 r / min and oscillation for 0.5-1.5 min.

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