Method for measuring concentrations of estradiol and non-binding oestrone in human plasma by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention discloses a method for measuring the concentration of estradiol and non-binding oestrone in human plasma through LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). The method comprises the following steps: carrying out pretreatment on a sample to be detected, and carrying out sample injection detection to obtain a response value; drawing a standard curve of estradiol and oestrone by using the concentration as a horizontal coordinate and the ratio of the standard curve sample to the internal standard response value as a vertical coordinate, and converting the response value of the sample to be detected into a concentration value according to the standard curve; the pretreatment comprises the following steps: taking to-be-detected plasma, adding an internal standard working solution, adding ethyl acetate for extraction, carrying out vortex mixing, then carrying out centrifugal treatment, taking supernatant, carrying out volatilization drying, adding a methanol-water mixed solution for redissolving, carrying out vortex uniform mixing, then carrying out centrifugation, and taking supernatant; the detection quantitation limit of the method is as follows: estradiol is 1pg / mL, and oestrone is 5pg / mL. According to the invention, efficient and accurate analysis of the content of estradiol with low concentration level and low non-binding estradiol in human plasma is realized.

Description

Technical Field

[0001] The present application relates to the field of trace detection, and in particular to a method for determining the concentration of estradiol and unbound estrone in human plasma by LC-MS / MS. Background Art

[0002] Estrogen is a female steroid hormone, mainly including estrone (E1), estradiol (E2) and estriol (E3). Its function is to promote the maturation of female sexual organs and the appearance of secondary sexual characteristics, and maintain normal sexual desire and reproductive function.

[0003] Estrone is widely present in women's bodies. It is the only estrogen that can still exist in women after menopause. Estradiol is a hormone mainly secreted by the ovaries. It is the main sex hormone for women of childbearing age and an important hormone for maintaining female characteristics and functions. At the same time, estradiol is very important for women's bone health. Estradiol imbalance can cause related gynecological diseases and even cancer. Estriol is produced by the placenta and has a very significant physiological significance mainly during pregnancy.

[0004] Currently, clinical estrogen testing mostly uses biochemical immunoassays, with estradiol as the main test item. However, most direct immunoassays can only detect concentrations as low as 30 pg / mL, which is difficult to meet the measurement needs of low-concentration groups such as children, menopausal women, and men.

[0005] Liquid chromatography-mass spectrometry combines the separation efficiency of high-performance liquid chromatography with the high sensitivity of mass spectrometry, and has potential for rapid separation and analysis of biological samples. However, estrogen exists in trace amounts in tissues and organisms, and its content in plasma is extremely low. The compound has low polarity, which results in low ionization efficiency in ESI and APCI sources, which greatly hinders the detection of liquid chromatography-mass spectrometry. In addition, the derivatization method used to improve sensitivity is not effective for estradiol detection, and may cause the hydrolysis of estrogen bound to proteins, which not only affects the accuracy of the results, but also has problems such as time-consuming and complicated operation. Summary of the invention

[0006] In order to achieve efficient and accurate analysis of the content of unconjugated estrone and low-concentration levels of estradiol in human plasma, the present application provides a method for determining the concentration of estradiol and unconjugated estrone in human plasma by LC-MS / MS.

[0007] The present application provides a method for determining the concentration of estradiol and unconjugated estrone in human plasma by LC-MS / MS, which comprises:

[0008] Perform pretreatment of the sample to be tested, inject the sample for testing, and obtain the response value;

[0009] Using the concentration as the horizontal axis and the ratio of the standard curve sample to the internal standard response value, the standard curve of estradiol and estrone is drawn, and the response value of the sample to be tested is converted into a concentration value according to the standard curve;

[0010] The response value of the target compound is corrected using the response value of the internal standard, and the corrected concentration value is calculated;

[0011] The pretreatment comprises: taking the plasma to be tested, adding the internal standard working solution, adding ethyl acetate for extraction, vortex mixing and then centrifuging, taking the upper layer liquid to evaporate, adding the methanol-water mixture to re-dissolve, vortex mixing and then centrifuging, and taking the supernatant;

[0012] The detection limits of the method are 1 pg / mL for estradiol and 5 pg / mL for estrone.

[0013] In any of the above technical solutions, the internal standard working solution contains isotopes of estradiol-d3 and estrone-d4, and the isotope internal standards are used to correct the influence of liquid chromatography-mass spectrometry instrument response and matrix interference.

[0014] In any of the above technical solutions, the internal standard working solution is a 50% methanol aqueous solution containing 0.1 ng / mL estradiol-d3 and 0.5 ng / mL estrone-d4.

[0015] In any of the above technical solutions, the target compounds are estradiol and estrone.

[0016] In any of the above technical solutions, the volume ratio of the plasma, internal standard working solution and ethyl acetate is 1:0.1-0.2:1-3.

[0017] It should be noted that the 50% methanol aqueous solution refers to a mixed solution of methanol and water in a volume ratio of 1:1. LC-MS / MS is a liquid chromatography-tandem mass spectrometry system, and its mass spectrometry equipment uses SCIEX tandem quadrupole mass spectrometry TripleQuad TM 5500, which combines the separation capabilities of ultra-performance liquid chromatography (UPLC) to achieve high-sensitivity and stability detection.

[0018] In any of the above technical solutions, the matrix of the standard curve samples is blank human serum from which endogenous substances have been removed; and the matrix of the quality control samples in the LC-MS / MS determination is plasma samples of menopausal women.

[0019] In any of the above technical solutions, the blank human serum is T3, T4 free Human Serum.

[0020] In any of the above technical solutions, the standard curve samples are prepared according to the following steps:

[0021] Estradiol and estrone stock solutions were prepared with DMSO at a concentration of 1 mg / mL.

[0022] Take the estradiol standard curve stock solution and the estrone standard curve stock solution, add the methanol-water mixture, and obtain a series of standard curve working solutions of estradiol and estrone with different concentrations;

[0023] Take blank human serum, add standard curve working solution and internal standard working solution, add ethyl acetate for extraction, vortex mix and centrifuge, take the upper liquid and evaporate, add methanol and water mixture to re-dissolve, vortex mix and centrifuge, and take the supernatant.

[0024] The present application adopts a simple and efficient liquid-liquid extraction method to pre-treat the plasma sample. The target compound is extracted from the plasma by adding ethyl acetate, and then centrifuged after vortex mixing, concentration and re-dissolution steps are performed to remove interfering substances, thereby improving the purity and concentration of the target compound in the sample to be tested, which is beneficial to improving the accuracy and precision of the test results.

[0025] In addition, the blank human serum used in this application is human serum from which endogenous thyroid hormones (T3 and T4) have been removed. As an alternative matrix, it eliminates the interference of endogenous substances, so that the standard curve can be more accurately quantified. More importantly, compared to using organic solvents as blank matrix, blank human serum still retains other biomolecules, such as proteins, lipids, carbohydrates, etc. These components are very similar to the matrix in the actual sample to be tested, and can better simulate the complexity of the real sample, reduce the influence of the matrix effect on the test results, and ensure that the standard curve sample and the actual sample have a similar environment during the ionization process, simulate the ionization interference in the actual sample, so as to facilitate correction and improve the accuracy of the quantitative results. The blank plasma of menopausal women is used as the quality control sample to measure the background value. The quality control sample is obtained by adding the quality control working solution to the blank plasma, and the quality control concentration value is obtained. The value of the background value is subtracted by the quality control concentration value, and the accuracy is calculated by dividing the theoretical value to ensure the accuracy of the detection.

[0026] In any of the above technical solutions, the mobile phase A of the liquid chromatography mobile phase in the detection is a 0.1-0.3 mmol / L ammonium fluoride aqueous solution, and the mobile phase B is a 0.1-0.3 mmol / L ammonium fluoride acetonitrile solution.

[0027] In any of the above technical solutions, the gradient elution concentration of the liquid chromatography in the detection is set to:

[0028] 0~0.5min: 60~70%A, 30~40%B;

[0029] 0.5~2.5min: 60~70%A→5~15%A, 30~40%B→85~95%B;

[0030] 2.5~3.0min: 5~15%A→5~15%A, 85~95%B→85~95%B;

[0031] 3.0~3.1min: 5~15%A→60~70%A, 85~95%B→30~40%B;

[0032] 3.1~4.0min: 60~70%A→60~70%A, 30~40%B→30~40%B.

[0033] Exemplarily, the gradient elution concentration is set as:

[0034] 0~0.5min: 65%A, 35%B;

[0035] 0.5~2.5min: 65%A→10%A, 35%B→90%B;

[0036] 2.5~3.0min: 10%A, 90%B;

[0037] 3.0~3.1min: 10%A→65%A, 90%B→35%B;

[0038] 3.1~4.0min: 65%A, 35%B.

[0039] In liquid chromatography, adding ammonium fluoride to the mobile phase can adjust its pH and interact with the target compound to reduce the adsorption or tailing of the compound on the chromatographic column, making the peak shape sharper and more symmetrical. This helps to improve the separation between estradiol and unconjugated estrone and between them and other impurities, thereby further improving the accuracy of the measurement results.

[0040] In the subsequent mass spectrometry detection, due to the use of an electrospray ion source for ionization, when a mobile phase with a certain concentration of electrolyte (ammonium fluoride) carries the target compound into the ion source, the electrolyte can assist the target compound in forming charged ions. In the negative ion mode, this promotion effect can form a certain ion pair with the target compound or change its surface charge distribution, making it easier for the target compound to form negative ions under the action of a high electric field, thereby improving the ionization efficiency and increasing the sensitivity of the detection.

[0041] In addition, the adjustment of the ratio of mobile phase A and B in the present application maintains a low acetonitrile ratio (35% B) at the beginning of the elution procedure (0-0.5min), so that the target compound is properly retained on the chromatographic column, thereby being separated from impurities with large polarity differences. As the elution process proceeds, the acetonitrile ratio gradually increases, so that the target compound can be eluted from the chromatographic column in sequence according to its distribution coefficient under the mobile phase system, which is beneficial to improving the accuracy of the analysis results.

[0042] In any of the above technical solutions, the column temperature of the liquid chromatography in the detection is 40-46°C.

[0043] In any of the above technical solutions, the chromatographic column of the liquid chromatography used for the detection is Waters ACQUITYUPLC BEH C18.

[0044] In any of the above technical solutions, the flow rate of the liquid chromatography for detection is 0.4 mL / min; the injection volume is 20 μL; and the temperature of the automatic sampler is 4° C.

[0045] In any of the above technical solutions, the electron source of the mass spectrometer in the detection is an electrospray ion source, in negative ion mode, using a multiple reaction monitoring mode, the scanning parameters are:

[0046] Estradiol: parent ion mass number is 271.050, product ion mass number is 145.000, declustering voltage is -180V, collision energy is -54V;

[0047] Estrone: parent ion mass number is 269.078, product ion mass number is 145.100, declustering voltage is -180V, collision energy is -52V;

[0048] Estradiol-d3: parent ion mass number is 274.072, product ion mass number is 145.200, declustering voltage is -235V, collision energy is -56V;

[0049] Estrone-d4: parent ion mass number is 273.035, product ion mass number is 147.000, declustering voltage is -180V, collision energy is -50V.

[0050] In any of the above technical solutions, the mass spectrometry ion source parameters in the detection are: Curtain Gas: 35 L / min; Collision Gas: 9; Ionspray Voltage: -4500 eV; Temperature: 550°C; Ion Source Gas1: 40 L / min; Ion Source Gas2: 60 L / min.

[0051] In summary, this application has the following beneficial effects:

[0052] The present invention provides a biological sample analysis method for simultaneously determining the concentration of human plasma estradiol and unbound estrone, and performs a joint analysis of estradiol and unbound estrone by an efficient, rapid and sensitive liquid phase-tandem mass spectrometry (LC-MS / MS) method. This method adopts a simple liquid-liquid extraction method to pre-treat the biological sample, has simple steps, is easy to operate, and does not require derivatization.

[0053] Estradiol and estrone are endogenous substances. The present invention uses blank human serum (T3, T4 free human serum) from which endogenous substances have been removed as a substitute matrix free of interference from endogenous substances for preparing blank samples, zero concentration samples and standard curve samples, achieving extremely low limits of quantification (estradiol: 1.00 pg / mL; estrone: 5.00 pg / mL), which can meet the detection needs of people with clinical low estrogen levels, especially the measurement needs of menopausal women and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is the blank human serum matrix spectrum.

[0055] Figure 2 It is the spectrum of the zero concentration sample.

[0056] Figure 3 This is the minimum quantification limit spectrum.

[0057] Figure 4 This is the highest limit of quantitation spectrum.

[0058] Figure 5 This is the spectrum of the subject 0.25 hours after taking the drug.

[0059] Figure 6 This is the spectrum 1.5 hours after the subject took the drug.

[0060] Figure 7 This is the spectrum of the subject 5 hours after taking the drug.

[0061] Figure 8 is a typical standard curve graph for estradiol.

[0062] Fig. 9 This is a typical standard curve for estrone. DETAILED DESCRIPTION

[0063] Example

[0064] Instruments and reagents in the embodiments: liquid spectrometer-mass spectrometer (Shimadzu LC-30AD+Sciex Triple Quad5500); centrifugal concentrator (Labconco CENTRIVAP); estradiol standard (China Food and Drug Inspection Institute); estrone standard (China Food and Drug Inspection Institute); estradiol-d3 (PANPHY); estrone-d4 (Bepure); ammonium fluoride (aladdin); methanol (Merck KGaA Co.); acetonitrile (Merck KGaA Co.); dimethyl sulfoxide (Macklin); ethyl acetate (aladdin); blank human serum from which endogenous substances are removed (T3, T4 free Human Serum purchased from Equitech-Bio, batch numbers: P18080101SCHS and P21181474); blank plasma and whole blood from healthy postmenopausal women were provided by Kangya Hospital, Yiyang, Hunan.

[0065] The same LC-MS / MS system can also be used.

[0066] Example 1

[0067] A method for determining the concentration of estradiol and unconjugated estrone in human plasma by LC-MS / MS comprises the following steps:

[0068] (1) Preparation of stock solution and internal standard working solution

[0069] Preparation of stock solutions: Use DMSO to prepare estradiol, estrone, estradiol-d3 and estrone-d4 stock solutions at a concentration of 1 mg / mL.

[0070] Preparation of internal standard working solution: Take 50 μL of estradiol-d3 stock solution and 250 μL of estrone-d4 stock solution, add 200 μL of methanol-water mixture (50 vol%), prepare a working solution containing estradiol-d3 100 μg / mL and estrone-d4 500 μg / mL, and prepare according to the table below to obtain a working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL. Take 1 mL of the working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL to a 200 mL volumetric flask, dilute with a methanol-water mixture (50 vol%) and adjust to the scale to obtain an internal standard working solution with an estradiol-d3 concentration of 0.1 ng / mL and an estrone-d4 concentration of 0.5 ng / mL.

[0071] (2) Liquid chromatography conditions

[0072] Chromatographic column: Waters ACQUITY UPLC BEH C18, (2.1×50mm, 1.7μm); flow rate: 0.4mL / min; injection volume: 20μL; column oven: 40℃; autosampler temperature: 4℃ mobile phase composition: mobile phase A: water (containing 0.2mmol ammonium fluoride), mobile phase B: acetonitrile (containing 0.2mmol ammonium fluoride). Gradient elution was used, and the elution program is shown in the table below.

[0073] Analysis time (min) Module Mobile phase B (%) Mobile phase A (%) 0.50 Pumps 35 65 2.50 Pumps 90 10 3.00 Pumps 90 10 3.10 Pumps 35 65 4.00 Controller Stop Stop

[0074] (3) Mass spectrometry conditions

[0075] Mass spectrometry analysis was performed using ESI source, negative ion, and MRM mode. The mass spectrometry ion source parameters and compound detection parameters are shown in the following table:

[0076] parameter Numeric Curtain Gas (L / min) 35 CollisionGas 9 IonsprayVoltage(eV) -4500 Temperature(℃) 550 IonSourceGas1(L / min) 40 IonSourceGas2(L / min) 60

[0077] The main scanning parameters of the analyte and internal standard are as follows:

[0078] Compound Q1Mass Q3Mass DP(V) EP(V) CE(V) CXP(V) Estradiol 271.050 145.000 -180 -10 -54 -11 Estradiol-d3 274.072 145.200 -235 -10 -56 -9 Estrone 269.078 145.100 -180 -10 -52 -11 estrone-d4 273.035 147.000 -180 -10 -50 -13

[0079] (4) Sample processing

[0080] Samples to be tested: Healthy postmenopausal women were used as subjects. Around 8:00 am on the day of administration, 1 mg of the test preparation estradiol valerate tablets were administered 30 minutes after a meal (starting from the first bite of food), all with 240 mL of warm water. About 6 mL of upper limb venous blood was collected 1 hour before administration, 0.5 hours before administration, before administration (0 hours), and 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 24 hours, 48 ​​hours, and 72 hours after administration, and placed in a blood collection tube containing K2EDTA anticoagulant as the plasma sample to be tested.

[0081] Take 500 μL of the plasma sample to be tested, add 50 μL of the internal standard working solution, add 1 mL of ethyl acetate, vortex mix, centrifuge at 1500 rcf for 10 min in a 4°C low-temperature centrifuge, take 400 μL of the upper layer liquid and place it in a centrifugal concentrator at 40°C, set it to evaporate for 30 min, add 50 μL of methanol and water mixture (50 vol%) to re-dissolve, vortex mix, centrifuge at 20000 rcf for 10 min in a 4°C low-temperature centrifuge, and take the supernatant for sampling and testing. The detection spectrum 0.25 h after administration is as follows Figure 5 As shown; 1.5h after administration, the detection spectrum is as follows Figure 6 The detection spectrum 5 hours after administration is shown as Figure 7 shown.

[0082] Example 2 (method selectivity study)

[0083] (1) Effect of matrix on analytes and internal standards: Take 500 μL of blank plasma from 6 different sources, add 50 μL of methanol-water mixture (50 vol%), add 1 mL of ethyl acetate, vortex mix, centrifuge at 1500 rcf for 10 min in a 4°C low-temperature centrifuge, take 400 μL of the upper layer, place in a centrifugal concentrator at 40°C, set for 30 min to evaporate, add 50 μL of methanol-water mixture (50 vol%) to re-dissolve, vortex mix, centrifuge at 20000 rcf for 10 min in a 4°C low-temperature centrifuge, and take the supernatant. 1 replicate was measured.

[0084] (2) Effect of internal standard on the analyte: Take 500 μL of T3, T4 free human serum, add 50 μL of internal standard working solution, add 1 mL of ethyl acetate, vortex mix, centrifuge at 1500 rcf for 10 min in a 4°C low-temperature centrifuge, take 400 μL of the upper layer liquid, place it in a centrifugal concentrator at 40°C, set it to evaporate for 30 min, add 50 μL of methanol and water mixture (50 vol%) to re-dissolve, vortex mix, centrifuge at 20000 rcf for 10 min in a 4°C low-temperature centrifuge, and take the supernatant. 1 replicate was measured in parallel.

[0085] (3) Effect of the analyte on the internal standard: Take 475 μL of T3, T4 free human serum, add 25 μL of ULOQ working solution (containing estradiol 2 ng / mL, estrone 10 ng / mL), add 50 μL of methanol-water mixture (50 vol%), add 1 mL of ethyl acetate, vortex mix, centrifuge at 1500 rcf for 10 min in a 4°C low-temperature centrifuge, take 400 μL of the upper layer liquid, place it in a centrifugal concentrator at 40°C, set it to evaporate for 30 min, add 50 μL of methanol-water mixture (50 vol%) to dissolve, vortex mix, centrifuge at 20000 rcf for 10 min in a 4°C low-temperature centrifuge, and take the supernatant. Perform the determination in parallel.

[0086] The results are shown in Table 1-1 and Table 1-2. The responses of the blank matrices from 6 different sources at the estradiol retention time were 143.9% to 340.1% of the responses in the LLOQ, and the responses at the internal standard retention time were 0.0% of the responses in the LLOQ; the responses at the estrone retention time were 172.8% to 429.8% of the responses in the LLOQ, and the responses at the internal standard retention time were 0.0% of the responses in the LLOQ. The responses of the T3, T4 free Human Serum matrix with internal standard added at the estradiol retention time were 0.0% of the responses in the LLOQ, and the responses at the estrone retention time were 0.0% of the responses in the LLOQ. The responses of the T3, T4 free Human Serum matrix with ULOQ concentrations of estradiol and estrone added at the internal standard retention time were 0.0% of the responses in the LLOQ.

[0087] Table 1-1 Selectivity Study (Estradiol)

[0088]

[0089] Table 1-2 Selectivity Study (Estrone)

[0090]

[0091]

[0092] Example 3 (Standard Curve)

[0093] Preparation of standard curve working solution: Take 200 μL of estradiol standard curve stock solution (1 mg / mL) and 1000 μL of estrone standard curve stock solution (1 mg / mL), add 800 μL of methanol-water mixture (50 vol%) to obtain a mixed standard solution with an estradiol concentration of 100,000 ng / mL and an estrone concentration of 500,000 ng / mL, and prepare the standard curve working solution according to the table below.

[0094]

[0095]

[0096] Preparation of internal standard working solution: Take 50 μL of estradiol-d3 stock solution and 250 μL of estrone-d4 stock solution, add 200 μL of methanol-water mixture (50 vol%), prepare a working solution containing estradiol-d3 100 μg / mL and estrone-d4 500 μg / mL, and prepare according to the following table to obtain a working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL. Take 1 mL of the working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL to a 200 mL volumetric flask, dilute with a methanol-water mixture (50 vol%) and adjust to the scale to obtain a working solution with an estradiol-d3 concentration of 0.1 ng / mL and an estrone-d4 concentration of 0.5 ng / mL.

[0097] Blank sample: Take 500μL of T3, T4 free Human Serum, add 50μL of methanol and water mixture (50vol%), add 1mL of ethyl acetate, vortex mix, centrifuge at 1500rcf for 10min in a 4℃ low-temperature centrifuge, take 400μL of the upper layer liquid and place it in a centrifugal concentrator at 40℃, set it for 30min to evaporate, add 50μL of methanol and water mixture (50vol%) to dissolve, vortex mix, centrifuge at 20000rcf for 10min in a 4℃ low-temperature centrifuge, and take the supernatant.

[0098] Zero concentration sample: Take 500μL of T3, T4 free human serum, add 50μL of internal standard working solution, add 1mL of ethyl acetate, vortex mix, centrifuge at 1500rcf for 10min in a 4℃ low-temperature centrifuge, take 400μL of the upper layer liquid and place it in a centrifugal concentrator at 40℃, set it for 30min to evaporate, add 50μL of methanol and water mixture (50vol%) to re-dissolve, vortex mix, centrifuge at 20000rcf for 10min in a 4℃ low-temperature centrifuge, and take the supernatant.

[0099] Standard curve sample: Take 475μL of T3, T4 free Human Serum, add 25μL of standard curve working solution, add 50μL of internal standard working solution, add 1mL of ethyl acetate, vortex mix, centrifuge at 1500rcf for 10min in a 4℃ low-temperature centrifuge, take 400μL of the upper layer liquid and place it in a centrifugal concentrator at 40℃, set it for 30min to evaporate, add 50μL of methanol and water mixture (50vol%) to re-dissolve, vortex mix, centrifuge at 20000rcf for 10min in a 4℃ low-temperature centrifuge, and take the supernatant.

[0100] The evaluation was performed by entering a standard curve at the beginning and end of each analytical batch. Using T3, T4free HumanSerum, serum samples containing estradiol (1.00, 2.00, 4.00, 5.00, 10.00, 20.00, 50.00, 100.00) pg / mL and estrone (5.00, 10.00, 20.00, 25.00, 50.00, 100.00, 250.00, 500.00) pg / mL were prepared. Two copies of each calibration concentration were prepared in parallel and measured after treatment.

[0101] Acceptance criteria: The calculated concentration of the calibration standard is within ±15% of the labeled value and within ±20% at the LLOQ. At least 75% of the calibration standards should meet the above criteria, and the LLOQ and ULOQ are not allowed to exceed the above criteria. The correlation coefficient R2 should be no less than 0.9800.

[0102] The standard curve parameters are summarized in Tables 2-1 to 2-2, the back-calculated concentrations of the standard curve are shown in Tables 3-1 to 3-2, and the typical calibration curve of estradiol is shown in Figure 8 , a typical calibration curve for estrone is shown in Fig. 9 The linear correlation coefficient (R2) of the calibration curve of estradiol was 0.9901-0.9979, all greater than 0.9800; the accuracy deviation % of each concentration of 10 batches was -0.9%-1.3%, and the %CV was 2.3%-10.0%; the linear correlation coefficient (R2) of the calibration curve of estrone was 0.9921-0.9998, all greater than 0.9800; the accuracy deviation % of each concentration of 10 batches was -1.5%-2.0%, and the %CV was 1.8%-4.5%. It shows that this method has good linearity when the concentration of estradiol is (1.00-100.00) pg / mL and the concentration of estrone is (5.00-500.00) pg / mL.

[0103] Table 2-1 Estradiol standard curve parameters

[0104] Standard: R of the standard curve fit 2 ≥0.9800, at least 75% of the samples have a calculated concentration within ±15% of the labeled value, and the lower limit of quantification is ±20%. Concentration unit: pg / mL

[0105]

[0106] Table 2-2 Standard curve parameters (estrone)

[0107] Standard: R of the standard curve fit 2 ≥0.9800, at least 75% of the samples have a calculated concentration within ±15% of the labeled value, and the lower limit of quantification is ±20%. Concentration unit: pg / mL

[0108]

[0109]

[0110] Table 3-1 Calculation of concentrations by standard curve (p-estradiol) Concentration unit: pg / mL

[0111]

[0112]

[0113] Table 3-2 Calculation of concentration by standard curve (estrone)

[0114] Concentration unit: pg / mL

[0115]

[0116] Example 4 (Precision and Accuracy)

[0117] Preparation of quality control working solution:

[0118] Take 200 μL of estradiol quality control stock solution (1 mg / mL) and 1000 μL of estrone quality control stock solution (1 mg / mL), add 800 μL of methanol-water mixture (50 vol%) to obtain a mixed standard solution with an estradiol concentration of 100,000 ng / mL and an estrone concentration of 500,000 ng / mL, and prepare the quality control working solution according to the table below.

[0119]

[0120] Preparation of internal standard working solution:

[0121] Take 50 μL of estradiol-d3 stock solution and 250 μL of estrone-d4 stock solution, add 200 μL of methanol-water mixture (50 vol%), prepare a working solution containing estradiol-d3 100 μg / mL and estrone-d4 500 μg / mL, and prepare according to the following table to obtain a working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL. Take 1 mL of the working solution with an estradiol-d3 concentration of 20 ng / mL and an estrone-d4 concentration of 100 ng / mL to a 200 mL volumetric flask, dilute with a methanol-water mixture (50 vol%) and adjust to the scale to obtain a working solution with an estradiol-d3 concentration of 0.1 ng / mL and an estrone-d4 concentration of 0.5 ng / mL.

[0122] Blank sample: Take 475 μL of blank plasma donated by menopausal women, add 25 μL of methanol-water mixture (50 vol%), add 50 μL of internal standard working solution, add 1 mL of ethyl acetate, vortex mix and centrifuge at 1500 rcf in a 4°C low-temperature centrifuge for 10 min, take 400 μL of the upper layer liquid and place it in a centrifugal concentrator at 40°C, set it for 30 min to evaporate, add 50 μL of methanol-water mixture (50 vol%) to re-dissolve, vortex mix and centrifuge at 20000 rcf in a 4°C low-temperature centrifuge for 10 min, take the supernatant for measurement, repeat 3 times, and set the average concentration as C1.

[0123] Accuracy and precision samples: Take 475 μL of blank plasma donated by menopausal women, add 25 μL of quality control working solution of LLOQ, QCL, QCM, and QCH, add 50 μL of internal standard working solution, add 1 mL of ethyl acetate, vortex mix and centrifuge at 1500 rcf in a 4°C low-temperature centrifuge for 10 min, take 400 μL of the upper layer liquid and place it in a centrifugal concentrator at 40°C, set it for 30 min to evaporate, add 50 μL of methanol and water mixture (50 vol%) to re-dissolve, vortex mix and centrifuge at 20000 rcf in a 4°C low-temperature centrifuge for 10 min, take the supernatant for determination, prepare 6 copies of each concentration in parallel, set the concentration as C2, examine 3 batches, and complete it in 3 days.

[0124] Acceptance criteria: Accuracy% = (C2-C1) / labeled value*100%; %CV was calculated using the (C2-C1) value at each concentration level.

[0125] Intra-batch accuracy and precision: The intra-batch accuracy mean should be within ±15% of the labeled value, and the LLOQ accuracy mean should be within ±20% of the labeled value. The intra-batch %CV should not exceed 15%, and the %CV of the LLOQ should not exceed 20%.

[0126] Inter-batch accuracy and precision: The inter-batch accuracy mean of at least 3 batches should be within ±15% of the labeled value, and the LLOQ accuracy mean should be within ±20% of the labeled value. The inter-batch %CV should not exceed 15%, and the %CV of the LLOQ should not exceed 20%.

[0127] The precision and accuracy results are shown in Tables 4-1 to 4-2. The intra-batch accuracy deviation % of estradiol at the quantitative lower limit is -6.7% to -3.3%, the intra-batch %CV does not exceed 16.7%, the inter-batch accuracy deviation % is -5.4%, the inter-batch %CV is 14.8%, the accuracy deviation does not exceed 20%, and the %CV does not exceed 20%, all of which meet the verification requirements; the intra-batch accuracy deviation % of estrone at the quantitative lower limit is -1.6% to 8.6%, the intra-batch %CV does not exceed 7.3%, the inter-batch accuracy deviation % is 4.5%, the inter-batch %CV is 6.9%, the accuracy deviation does not exceed 20%, and the %CV does not exceed 20%, all of which meet the verification requirements. The intra-batch accuracy deviation % of estradiol is between -2.6% and 5.7%, the intra-batch %CV does not exceed 10.2%, the inter-batch accuracy deviation % is 0.6% to 1.2%, and the inter-batch %CV does not exceed 8.4%. The accuracy deviation does not exceed 15%, and the %CV does not exceed 15%, all of which meet the verification requirements. The intra-batch accuracy deviation of estrone is -8.3% to 9.0%, the intra-batch %CV does not exceed 7.0%, the inter-batch accuracy deviation is -0.1% to 2.7%, and the inter-batch %CV does not exceed 7.5%. The accuracy deviation does not exceed 15%, and the %CV does not exceed 15%, which meets the verification requirements.

[0128] Table 4-1 Precision and accuracy of intra-batch and inter-batch sample testing (estradiol)

[0129]

[0130]

[0131]

[0132] *Outliers are not included in the calculation.

[0133] Table 4-2 Precision and accuracy of intra-batch and inter-batch sample testing (estrone)

[0134]

[0135]

[0136] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for determining the concentration of estradiol and unbound estrone in human plasma by LC-MS / MS, characterized in that: include: Perform pretreatment of the sample to be tested, inject the sample for testing, and obtain the response value; Using the concentration as the horizontal axis and the ratio of the standard curve sample and the internal standard response value as the vertical axis, the standard curve of estradiol and estrone is drawn, and the response value of the sample to be tested is converted into a concentration value according to the standard curve; The pretreatment comprises: taking the plasma to be tested, adding the internal standard working solution, adding ethyl acetate for extraction, vortex mixing and then centrifuging, taking the upper layer liquid to evaporate, adding the methanol-water mixture to re-dissolve, vortex mixing and then centrifuging, and taking the supernatant; The detection limits of the method are 1 pg / mL for estradiol and 5 pg / mL for estrone.

2. The method according to claim 1, characterized in that: The internal standard working solution is a 50% methanol aqueous solution containing 0.1 ng / mL estradiol-d3 and 0.5 ng / mL estrone-d4.

3. The method according to claim 1, characterized in that The matrix of the standard curve samples is blank human serum from which endogenous substances have been removed.

4. The method according to claim 3, characterized in that: The blank human serum is T3, T4 free HumanSerum.

5. The method according to claim 1, characterized in that The standard curve samples were prepared according to the following steps: Estradiol and estrone stock solutions were prepared with DMSO at a concentration of 1 mg / mL; Take the estradiol standard curve stock solution and the estrone standard curve stock solution, add the methanol-water mixture, and obtain a series of standard curve working solutions of estradiol and estrone with different concentrations; Take blank human serum, add standard curve working solution and internal standard working solution, add ethyl acetate for extraction, vortex mix and centrifuge, take the upper liquid and evaporate, add methanol and water mixture to re-dissolve, vortex mix and centrifuge, and take the supernatant.

6. The method according to claim 1, characterized in that In the detection, the mobile phase A of the liquid chromatography mobile phase is a 0.1-0.3 mmol / L ammonium fluoride aqueous solution, and the mobile phase B is a 0.1-0.3 mmol / L ammonium fluoride acetonitrile solution.

7. The method according to claim 1, characterized in that The gradient elution concentration of the liquid chromatography in the detection is set as: 0~0.5min: 60~70%A, 30~40%B; 0.5~2.5min: 60~70%A→5~15%A, 30~40%B→85~95%B; 2.5~3.0min: 5~15%A→5~15%A, 85~95%B→85~95%B; 3.0~3.1min: 5~15%A→60~70%A, 85~95%B→30~40%B; 3.1~4.0min: 60~70%A→60~70%A, 30~40%B→30~40%B.

8. The method according to claim 1, characterized in that: The column temperature of the liquid chromatography in the detection is 40-46°C.

9. The method according to claim 1, characterized in that: The chromatographic column of the liquid chromatography in the detection is Waters ACQUITY UPLC BEH C18; preferably, the flow rate is 0.4 mL / min; and the injection volume is 20 mL.

10. The method according to claim 1, characterized in that The electron source for mass spectrometry analysis in the detection is an electrospray ion source, in negative ion mode, using multiple reaction monitoring mode, the scanning parameters are: Estradiol: parent ion mass number is 271.050, product ion mass number is 145.000, declustering voltage is -180V, collision energy is -54V; Estrone: parent ion mass number is 269.078, product ion mass number is 145.100, declustering voltage is -180V, collision energy is -52V; Estradiol-d3: parent ion mass number is 274.072, product ion mass number is 145.200, declustering voltage is -235V, collision energy is -56V; Estrone-d4: parent ion mass number is 273.035, product ion mass number is 147.000, declustering voltage is -180V, collision energy is -50V.

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