LC-MS / MS method for rapidly quantifying testosterone concentration in serum

Abstract

The invention belongs to the field of in-vivo quantitative drug concentration analysis, and particularly discloses an LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) method for rapidly quantifying testosterone concentration in serum, which comprises the following steps: (1) establishing a substitute isotope testosterone d3 standard curve; and (2) determining the testosterone content in the serum sample to be detected. The serum sample is pretreated through a magnetic bead adsorption method, under the cooperation of chromatography and mass spectrometry conditions, the content of testosterone in the blood sample can be accurately and quantitatively detected only through a trace amount of the serum sample (100 microliters), the sensitivity is high, the detection limit is low, and pg / mL-level total testosterone in the serum can also be accurately detected.

Description

Technical Field

[0001] The invention relates to the field of in vivo quantitative drug concentration analysis, and in particular to an LC-MS / MS method for rapid quantitative analysis of testosterone concentration in serum. Background Art

[0002] Testosterone, also known as testosterone, testosterone or testosterone, is a steroid hormone from male hormones. Testosterone is mainly secreted by male testicles and female ovaries, and a small part is secreted by the adrenal glands. Testosterone has the functions of maintaining muscle strength and quality, maintaining bone density and strength, refreshing and improving physical fitness. Testosterone in serum affects many body systems and functions, including: blood production, calcium balance in the body, bone mineralization, lipid metabolism, sugar metabolism and prostate growth. Whether it is men or women, testosterone in serum has an important impact on health. The determination of testosterone in serum can help diagnose and treat a variety of endocrine and metabolic diseases, and can also help evaluate the effectiveness of drug treatment. It can also strictly monitor whether athletes abuse testosterone to ensure fair competition.

[0003] Traditionally, radioimmunoassay or ELISA methods are used to measure testosterone in serum. These methods have the problem of false positive interference from antigens and antibodies, and inaccurate determination of hormones at low concentration levels. LC-MS has the advantages of strong specificity, high detection sensitivity, and suitability for high-throughput determination. Currently, there are studies reporting the use of LC-MS to quantify testosterone concentration in serum. For example, Chu Yang et al. used liquid-liquid extraction to extract testosterone from serum in <High Performance Liquid Chromatography-Tandem Mass Spectrometry for Determination of Endogenous Testosterone Content in Human Serum>, and then used LC-MS to measure testosterone. This method has low sensitivity, and the testosterone content in women and children is generally low. The testosterone content in men after drug castration for prostate cancer is usually very low, and can reach the lowest level of pg / ml. At this time, accurate determination of testosterone cannot be achieved; Patent CN 116046937A discloses a high performance liquid chromatography tandem mass spectrometry detection method for free testosterone, which is derived from testosterone in serum and detected by liquid-liquid extraction. Although it has high sensitivity and can detect pg / ml level free testosterone in serum, the amount of serum used is large, which will increase discomfort for patients who need frequent monitoring, and because only the testosterone concentration of the free part of total testosterone can be detected, when diagnosing and treating low testosterone, reproductive system diseases, endocrine dysfunction and other diseases, it is necessary to assess the level of androgen in the body by detecting the total testosterone concentration in serum, and this method cannot be used for detection. When using animal model mice and rats to study testosterone-related diseases, the currently disclosed liquid-mass spectrometry method has a large demand for serum detection at one time, which exceeds the range that it can adapt to, resulting in death of the model before the test cycle is completed, and the disease research is forced to terminate. These problems in the detection method of testosterone concentration in serum in clinical applications and drug animal experiments have made it an urgent need to develop a rapid, highly sensitive and low serum demand testosterone LC-MS quantitative detection method. Summary of the invention

[0004] To solve the above problems, the present invention provides a LC-MS / MS analysis method for rapid quantitative analysis of testosterone concentration in serum, which comprises the following steps:

[0005] (1) Establishment of testosterone-d3 standard curve

[0006] a. Preparation of working solution for series concentration standard curve: take testosterone-d3 standard substance, add acetonitrile to dissolve, and dilute the dissolved solution into series concentration by adding methanol solution; respectively take solutions of series concentration, add matrix, water and internal standard solution to mix, extract the mixed solution by magnetic bead adsorption method, and collect the eluate after extraction as the working solution for series concentration standard curve;

[0007] The steps of the magnetic bead adsorption method are:

[0008] The mixed solution is loaded onto the balanced magnetic bead solution, and then washed with an aqueous solution containing 0.05-0.15% formic acid and 30-40% methanol, and an aqueous solution containing 0.05-0.15% ammonia and 30-40% methanol, and then eluted with 70-90% methanol solution, and the eluate is collected;

[0009] b. Pipette a series of concentration standard working solutions and inject them into the LC-MS / MS instrument to measure the peak area. Draw a testosterone-d3 standard curve with the ratio of the peak area of ​​testosterone-d3 to the internal standard as the ordinate and the concentration of the testosterone-d3 standard substance as the abscissa;

[0010] The chromatographic conditions were as follows:

[0011] Chromatographic column: Octyl bonded silica gel as filler; acetic acid-ammonium acetate aqueous solution as mobile phase A, formic acid methanol as mobile phase B, gradient elution program:

[0012] Time / min Mobile phase A / % Mobile phase B / % 0.00 55 45 0.50 55 45 2.00 2 98 3.00 2 98 3.10 55 45 4.00 55 45

[0013] Mass spectrometry conditions: the ion source was electrospray ionization source; the mass spectrometry detection mode was multiple ion reaction monitoring;

[0014] (2) Determination of testosterone content in the sample to be tested:

[0015] c. Preparation of biological samples

[0016] Take a serum sample to be tested, add water and internal standard solution to mix, extract the mixed solution according to the same magnetic bead adsorption method in step a, collect the eluate after extraction, and obtain a biological sample;

[0017] d. Determination of biological samples

[0018] Take a biological sample, inject it into the LC-MS / MS instrument for detection, and detect it under the same conditions as step b, and obtain the testosterone content in the serum sample to be tested according to the standard curve of step (1).

[0019] Furthermore, in the content determination process of step (2), a quality control procedure is also included, which specifically comprises the following steps: taking a matrix, adding water and an internal standard solution in a volume ratio of 100:200:10 and mixing evenly; taking a testosterone-d3 standard substance and a testosterone standard substance respectively, adding acetonitrile to dissolve them; diluting the dissolved solution with a methanol solution to the same concentration and then mixing evenly with the matrix, water and the internal standard solution in a volume ratio of 10:90:200:10; extracting the mixed solution according to the same magnetic bead adsorption method as in step a, collecting the extracted eluate, injecting it into an LC-MS / MS instrument, and monitoring the interference in the determination process;

[0020] The concentration of the dissolving solution is 1.00 mg / mL, and the dissolving solution is diluted to a solution with a concentration of 0.1 to 100 ng / mL.

[0021] Furthermore, the volume ratio of the series concentration solution, matrix, water and internal standard solution in step a is 10:90:200:10; the concentration of testosterone-d3 in the series concentration solution is 0.1-100 ng / mL.

[0022] Furthermore, the matrix is ​​rat blank serum.

[0023] Furthermore, the steps of the magnetic bead adsorption method in step a) are:

[0024] After the magnetic bead solution is mixed and balanced with the balance solution, the sample mixture is adsorbed for 50-70 seconds, and then an aqueous solution containing 0.1% formic acid and 35% methanol, and an aqueous solution containing 0.1% ammonia and 35% methanol are added in sequence for elution, and finally an 80% methanol aqueous solution is added for elution, and the eluate is collected;

[0025] The volume ratio of the magnetic bead solution, the balance solution, the mixed solution, the aqueous solution containing 0.1% formic acid and 35% methanol, the aqueous solution containing 0.1% ammonia water and 35% methanol, and the 80% methanol solution is 200 μL: 500 μL: 310 μL: 500 μL: 500 μL: 160 μL;

[0026] The magnetic bead solution is an aqueous solution containing 5 mg / mL hydrophobic magnetic polymer microspheres and 30% methanol;

[0027] The balance solution is a 5% methanol aqueous solution;

[0028] The mixing equilibrium time is 20 s, the adsorption temperature is 45° C., the time is 60 s, the elution time is 20 s, the elution temperature is 45° C., and the time is 60 s.

[0029] Furthermore, when the steps of the magnetic bead adsorption method are performed in the sample pretreatment system MIP96, the speed gear of mixing balance, adsorption, elution and elution is 2 gears.

[0030] Furthermore, the internal standard solution is acetonitrile containing testosterone-d5 standard substance, which is diluted with methanol solution to a solution with a concentration of 1.00 ng / mL to 10.0 ng / mL.

[0031] Furthermore, in the chromatographic conditions of step b, the chromatographic column is ACQUITY UPLC BEH C8 1.8 μm 2.1×50 mm Column, the injection volume is 5 μL, the column temperature is 40° C., and the flow rate is 0.6 ml / min.

[0032] Furthermore, the mobile phase A is 0.1% acetic acid-2 mM ammonium acetate aqueous solution; and the mobile phase B is 0.1% formic acid methanol.

[0033] Furthermore, in the mass spectrometry conditions of step b, curtain gas pressure (CUR): 35 psi; collision gas pressure (CAD): 9.00 psi; ionization voltage (IS): 5500 V; nebulization temperature (TEM): 500° C.; nebulization gas pressure 1 (GS1): 55 psi; nebulization gas pressure 2 (GS2): 55 psi; scanning time: 4.0 min; declustering voltage (DP): 100 V, entrance voltage EP: 10 V, collision voltage (CXP) 11 V.

[0034] Furthermore, in the mass spectrometry conditions, the testosterone quantitative ion pair is m / z 289.2→m / z 97.1, and the collision energy (CE) is 27 eV; the testosterone qualitative ion pair is m / z 289.2→m / z 109.0, and the collision energy (CE) is 33 eV; the testosterone-d3 quantitative ion pair is m / z 292.2→m / z 97.1, and the collision energy (CE) is 25 eV, and the testosterone-d5 quantitative ion pair is m / z294.2→m / z100.0, and the collision energy (CE) is 30 eV.

[0035] The LC-MS / MS quantitative analysis method for testosterone concentration in serum of the present invention performs pretreatment on serum samples by a specific magnetic bead adsorption method, and under the cooperation of specific chromatographic mass spectrometry conditions, it is possible to accurately and quantitatively detect the testosterone content in a blood sample using only a trace amount of serum sample (100 μL), and the method has high sensitivity and a low detection limit, so that pg / ml level total testosterone in serum can also be measured, and the concentration variation range between the lowest concentration point and the highest concentration point of the standard curve can reach 1000 times, ensuring that when performing testosterone quantitative analysis, no matter the concentration of the blood sample is high or low, it can be performed within the linear dynamic region of LC-MS / MS, thereby ensuring the reliability and accuracy of the analysis result.

[0036] Since the standard curve used in the quantitative testosterone process is drawn with a testosterone-d3 solution, testosterone-d3 does not exist in the actual blood sample. In order to accurately quantify the testosterone in the serum by the standard curve drawn with a testosterone-d3 solution, it is necessary to make the chromatographic mass spectrometric behavior of testosterone-d3 and testosterone on the chromatogram consistent and the response consistent. For this reason, the present invention explores the chromatographic conditions and mass spectrometric conditions of serum samples treated by the magnetic bead adsorption method, and finds that when testosterone-d3 and testosterone enter the mass spectrometer after chromatographic separation, the declustering voltage (DP) and collision energy (CE) in the mass spectrometric conditions need to be under specific parameters, so that the preparation of the standard curve can be achieved by using the testosterone-d3 standard substance instead of the testosterone to be tested, thereby accurately quantifying the total testosterone in the test serum.

[0037] The LC-MS / MS quantitative analysis method of testosterone concentration in serum of the present invention provides a new method for the pharmacokinetic study of total testosterone, avoiding the problem that when using LC-MS / MS to quantify the total testosterone concentration in serum, it is impossible to accurately quantify the pg / ml level testosterone with trace serum. At the same time, the present invention uses high-throughput magnetic extraction to pre-treat the sample, which meets the requirements of the existing automated pre-treatment system. With its cooperation, it can be independent of manual labor and can complete the pre-treatment of the sample in a short time, with low cost. The pre-treated sample solution can achieve accurate quantification in a wide range of 10.0 to 10000 pg / mL in the serum under specific chromatographic mass spectrometry conditions. It not only meets the needs of animal experimental research, but also meets the needs of clinical detection, and has practical promotion and application value.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Chromatogram of testosterone in blank sample;

[0041] Figure 2 Chromatogram of testosterone-d3 in blank sample;

[0042] Figure 3 Chromatogram of testosterone-d5 in blank sample;

[0043] Figure 4 Chromatogram of testosterone-d3 in the sample with lower limit of quantification;

[0044] Figure 5 Chromatogram of testosterone-d5 in the sample with lower limit of quantification;

[0045] Figure 6 Chromatogram of testosterone-d3 in the upper limit of quantification sample

[0046] Figure 7 Chromatogram of testosterone-d3 in residual samples;

[0047] Figure 8 Typical standard curve for testosterone-d3 in rat serum. DETAILED DESCRIPTION

[0048] The reagents and equipment used in the specific embodiments of the present invention can be obtained through commercial purchase.

[0049] Example 1, Determination of testosterone concentration in serum

[0050] (I) Preparation of solution

[0051] Testosterone-d3 stock solution: weigh 1 mg of testosterone-d3 standard substance into a polypropylene tube, add acetonitrile to prepare a 1.00 mg / mL testosterone-d3 stock solution;

[0052] Testosterone stock solution: Take 1 mg of testosterone standard substance in a polypropylene tube, add acetonitrile, and prepare a 1.00 mg / mL testosterone stock solution;

[0053] Internal standard working solution: Take 1 mg of testosterone-d5 standard substance in a polypropylene tube, add acetonitrile to prepare a 1.00 mg / mL internal standard stock solution; take the internal standard stock solution and dilute it with 45% methanol aqueous solution to a 1.00 ng / mL internal standard solution;

[0054] Magnetic bead solution: Take hydrophobic (RPP) magnetic polymer microspheres, add 30% methanol aqueous solution, and prepare 5 mg / mL as magnetic bead solution;

[0055] Balance solution: Take methanol and add water to prepare a 5% methanol-water solution as the balance solution;

[0056] Eluent 1: Take formic acid and methanol, add water to prepare an aqueous solution containing 0.1% formic acid and 35% methanol as eluent 1;

[0057] Eluent 2: Take ammonia water and methanol, add water to prepare an aqueous solution containing 0.1% ammonia water and 35% methanol as eluent 2;

[0058] Eluent: Take methanol and add water to make 80% methanol-water solution as eluent;

[0059] (II) Establishment of testosterone-d3 standard curve

[0060] a. Preparation of a series of concentration standard curve working solutions and a series of concentration quality control solutions:

[0061] Take the testosterone-d3 stock solution and dilute it with 45% methanol aqueous solution to prepare a series of concentration standard curve solutions with concentrations of 100 ng / mL, 80.0 ng / mL, 20.0 ng / mL, 8.00 ng / mL, 2.00 ng / mL, 0.800 ng / mL, 0.200 ng / mL and 0.100 ng / mL;

[0062] Take 10 μL of the series concentration standard curve solution, add 90 μL of blank matrix rat blank serum and mix well, then add 200 μL of pure water and 10 μL of internal standard working solution, load the mixed solution into the sample pretreatment system MIP96 for high-throughput magnetic extraction, and collect the eluate after extraction as the series concentration standard curve working solution;

[0063] The working procedure of the sample pretreatment system MIP96 is:

[0064] 200 μL magnetic bead solution was mixed at the speed of 2 gear for 20 s, 500 μL balancing solution was added and mixed and balanced at the speed of 2 gear for 20 s, 310 μL mixed solution was loaded, adsorbed at 45°C at the speed of 2 gear for 60 s, 500 μL eluent 1 was added and mixed and eluted at the speed of 2 gear for 20 s, 500 μL eluent 2 was added and mixed and eluted at the speed of 2 gear for 20 s, 160 μL eluent was added and mixed and eluted at 45°C and the speed of 2 gear for 60 s, and the eluate was collected;

[0065] b. Pipette the working solution of the series concentration standard curve and inject it into the LC-MS / MS instrument to determine the peak area. The testosterone-d3 concentration of the working solution of the series concentration standard curve is used as the horizontal axis (X), and the peak area ratio of testosterone-d3 to the internal standard is used as the vertical axis (Y). The weighted least square method (W = 1 / X 2 ) to perform regression calculation and obtain a linear regression equation, which is the testosterone-d3 standard curve;

[0066] The chromatographic conditions were as follows:

[0067] The chromatographic column was ACQUITY UPLC BEH C8 1.8μm 2.1*50mm Column; the injection volume was 5μL; the column temperature was 40°C, the flow rate was 0.6ml / min; 0.1% acetic acid-2mM ammonium acetate aqueous solution was used as mobile phase A, 0.1% formic acid methanol was used as mobile phase B, and the gradient elution program was as follows:

[0068] Time / min Mobile phase A / % Mobile phase B / % 0.00 55 45 0.50 55 45 2.00 2 98 3.00 2 98 3.10 55 45 4.00 55 45

[0069] Mass spectrometry conditions: Ion source: electrospray ionization source;

[0070] Mass spectrometry detection mode (Scan Type): MRM;

[0071] Curtain air pressure (CUR): 35psi;

[0072] Collision gas pressure (CAD): 9.00 psi;

[0073] Ionization voltage (IS): 5500V:

[0074] Atomization temperature (TEM): 500°C;

[0075] Atomizing gas pressure 1 (GS1): 55psi;

[0076] Atomizing gas pressure 2 (GS2): 55psi;

[0077] Scan time: 4.0min;

[0078] Declustering voltage (DP): 100 V, entrance voltage (EP): 10 V, collision voltage (CXP) 11 V; testosterone quantitative ion pair m / z 289.2→m / z 97.1, collision energy (CE): 27 eV, testosterone qualitative ion pair m / z 289.2→m / z 109.0, collision energy (CE): 33 eV; testosterone-d3 quantitative ion pair m / z 292.2→m / z 97.1, collision energy (CE): 25 eV, testosterone-d5 quantitative ion pair

[0079] m / z294.2→m / z100.0, collision energy (CE): 30 eV;

[0080] (III) Determination of testosterone content in the sample to be tested

[0081] c. Preparation of biological samples, blank samples and quality control samples

[0082] Take 100 μL of serum sample into a 1.5 mL polypropylene centrifuge tube, add 200 μL of pure water and 10 μL of internal standard solution to mix, load the mixed solution into the sample pretreatment system MIP96, perform high-throughput magnetic extraction according to the same working procedure as step a), and collect the eluate to obtain the biological sample;

[0083] Take 100 μL of blank matrix into a 1.5 mL polypropylene centrifuge tube, add 200 μL of pure water and 10 μL of internal standard solution and mix, load the mixed solution into the sample pretreatment system MIP96, perform high-throughput magnetic extraction according to the same working procedure as step a), and collect the eluate to obtain a blank sample;

[0084] Take testosterone-d3 stock solution and testosterone stock solution respectively, add 45% methanol water to dilute to the same concentration of 5.00 ng / mL quality control working solution, take 10 μL of quality control working solution respectively, add 90 μL of blank matrix rat blank serum to mix, then add 200 μL of pure water and 10 uL of internal standard solution to mix, load the mixed solution on the sample pretreatment system MIP96, perform high-throughput magnetic extraction according to the same working procedure as step a), and collect the eluate to obtain the quality control sample;

[0085] d. Determination of biological samples

[0086] Take 5 μL of blank sample, quality control sample and biological sample respectively, inject into LC-MS / MS instrument for detection, and detect under the same conditions as step b, and use blank sample to monitor whether there is contamination in the operation process; use quality control sample to monitor whether the response of instrument to testosterone-d3 and testosterone is consistent, and obtain the testosterone content in the biological sample according to the standard curve of step (ii).

[0087] The beneficial effects of the present invention are described below through test examples.

[0088] Test Example 1 Methodological Verification of the Invention

[0089] 1. Instruments and chromatography-mass spectrometry conditions

[0090] 1.1 Main instruments and equipment

[0091]

[0092] 1.2 Chromatographic conditions

[0093]

[0094] Gradient elution program

[0095] Time(min) Flow rate (ml / min) B.Conc(%) 0.00 0.6 45 0.50 0.6 45 2.00 0.6 98 3.00 0.6 98 3.10 0.6 45 4.00 0.6 45

[0096] 1.3 Mass spectrometry conditions

[0097] Ion source parameters:

[0098] Parameter name Parameter settings Ion Source Electrospray ionization (ESI) Polarity Positive Scan Type MRM Curtain Gas(CUR) 35 Collision Gas(CAD) 9.00 IonSpray Voltage(IS) 5500 Temperature(TEM) 500 Ion Source Gas 1(GS1) 55 Ion Source Gas 2(GS2) 55 Duration 4.0min

[0099] Compound parameters:

[0100]

[0101]

[0102] 2 Preparation of stock solution, working solution and samples

[0103] 2.1 Reference Material Information

[0104] type name source Object to be tested Testosterone Beijing Manhag Biotechnology Co., Ltd. Object to be tested Testosterone-D3 Beijing Manhag Biotechnology Co., Ltd. Internal Standard Testosterone-D5 Beijing Manhag Biotechnology Co., Ltd.

[0105] 2.2 Preparation of stock solution

[0106] 2.2.1 Testosterone stock solution

[0107] Preparation of testosterone stock solution with a concentration of about 1.00 mg / mL: weigh about 1 mg of testosterone standard substance into a polypropylene tube, add a certain volume of acetonitrile to prepare 1.00 mg / mL as quality control stock solution, and store the stock solution in a polypropylene tube at 2-8°C.

[0108] 2.2.2 Testosterone-D3 stock solution

[0109] Preparation of testosterone-d3 stock solution with a concentration of about 1.00 mg / mL: weigh about 1 mg of testosterone-d3 standard substance into a polypropylene tube, add a certain volume of acetonitrile to prepare 1.00 mg / mL. Weigh two portions, one as the standard curve stock solution and the other as the quality control stock solution. The stock solution is stored in a polypropylene tube at 2-8°C.

[0110] 2.2.3 Internal Standard (IS) Stock Solution

[0111] Preparation of internal standard stock solution with a concentration of about 1.00 mg / mL: weigh about 1 mg of testosterone-d5 standard substance into a polypropylene tube, add a certain volume of acetonitrile to prepare 1.00 mg / mL. The stock solution is stored in a polypropylene tube at 2-8°C.

[0112] 2.3 Preparation of working solution

[0113] Using 45% methanol aqueous solution as the diluent, the standard curve stock solution was diluted stepwise in a polypropylene centrifuge tube to obtain a series of standard curve working solutions of 10000 (standard curve intermediate working solution), 100, 80.0, 20.0, 8.00, 2.00, 0.800, 0.200, and 0.100 ng / mL; the quality control stock solution was diluted stepwise to obtain a series of quality control working solutions of 10000 (quality control intermediate working solution), 75.0, 5.00, 0.250, and 0.100 ng / mL; the internal standard stock solution was diluted stepwise to obtain 10000 (internal standard intermediate working solution), 10.0 ng / mL (internal standard intermediate working solution),

[0114] 1.00 ng / mL series of internal standard working solutions. The internal standard working solutions were stored in polypropylene tubes at 2-8°C and used at 10-30°C.

[0115] 2.4 Preparation of standard curve and quality control samples

[0116] According to the ratio of blank matrix to working solution of 90:10 (v / v), take rat blank serum into a polypropylene centrifuge tube, add standard curve or quality control working solution, and vortex to mix.

[0117] 3. Sample processing

[0118] (1) All matrix samples were thawed at 10-30°C;

[0119] (2) Add samples to the sample pretreatment system MIP96 according to the following table:

[0120]

[0121] *Magnetic bead type: hydrophobic (RPP) magnetic polymer microspheres. Batch number: 20240222. Source: Nanjing ADM Technology Co., Ltd.

[0122] (3) Set up the sample pretreatment system according to the following procedure*:

[0123]

[0124] *Brand: Nanjing ADM Technology Co., Ltd.;

[0125] (4) Place the 96-well plate treated by the sample pretreatment system on the magnetic plate, draw 100 μL of the supernatant from the “elution” well into the injection vial, and inject for analysis.

[0126] (5) Each analytical batch needs to prepare two quality control medium concentration samples containing testosterone and testosterone-d3, and three zero concentration samples (containing only internal standards but not the analyte) to prevent changes in the instrument status from affecting testosterone detection and to ensure the consistency of the responses of the analyte testosterone (minus background) and the surrogate analyte testosterone-d3. If the difference in the peak area of ​​testosterone-d3 and testosterone in the quality control sample exceeds 5%, adjustments are made based on the original declustering voltage and testosterone quantitative ion pair collision energy and testosterone-d3 quantitative ion pair collision energy to ensure that the difference in the peak area of ​​testosterone-d3 and testosterone does not exceed 5%, thereby ensuring that the responses of testosterone and testosterone-D3 are consistent during the detection process.

[0127] 4. Methodological Validation Results

[0128] 4.1 Selectivity

[0129] Six blank serum samples from rats of different origins were prepared respectively, and six quantitative lower limit samples were prepared in parallel to investigate the interference of blank serum on testosterone-d3 and internal standard; six zero concentration samples (containing only internal standard but not analyte) were prepared in parallel to investigate the interference of internal standard on testosterone and testosterone-d3.

[0130] Prepare 6 serum samples with the upper limit of quantification containing only testosterone-d3 (without internal standard) in parallel to investigate the interference of testosterone-d3 on the internal standard. Prepare 6 serum samples with the upper limit of quantification containing only testosterone (without internal standard) in parallel to investigate the interference of testosterone on the internal standard. The peak area of ​​the analyte measured by the above blank and zero concentration samples should be compared with the mean peak area of ​​the analyte with the lower limit of quantification, and there should be no obvious (<20% peak area of ​​the analyte) interference, which does not affect the determination of testosterone-d3. The peak area of ​​the internal standard measured by the blank and upper limit of quantification samples should be compared with the mean peak area of ​​the internal standard of the LLOQ sample, and there should be no obvious (<5% peak area of ​​the internal standard) interference.

[0131] Peak purity was used to investigate the selectivity of the analyte, and two ion channels of the analyte were selected, of which the peak with the larger peak area was the quantitative peak and the other was the qualitative peak. Six blank serum samples (non-hemolytic and non-hyperlipidemic) were prepared in parallel. After the blank samples were extracted, they were compared with the pure solution of QC M concentration. The deviation of the mean retention time of the chromatographic peak was within ±2%. At the same time, the relative deviation of the ratio of the qualitative peak to the quantitative peak of the blank sample and the QC M sample met the requirements of the table below, which can prove that the chromatographic peak of the blank matrix is ​​produced by endogenous analytes, and the interference of other co-eluting substances in the blank matrix is ​​excluded.

[0132] Table 1 Relative deviation standard of the ratio of qualitative peak to quantitative peak

[0133] Qualitative / quantitative peak area ratio (%) ＞50 20~50(inclusive) 10~20(inclusive) ≤10 Relative deviation (%) ±20 ±25 ±30 ±50

[0134] The results show that the interference of blank serum from 6 different sources on testosterone-d3 is ≤14.5%, the interference on internal standard is ≤1.5%, the interference of internal standard on testosterone-d3 is ≤8.1%, the interference of testosterone-d3 on internal standard is ≤3.4%, and testosterone has no interference on internal standard. All meet the requirements (Table 2-3), and the typical chromatograms are shown in Figure 1 to Figure 7 .

[0135] Table 2 Interference evaluation of each sample on testosterone-d3

[0136]

[0137] Table 3 Interference evaluation of each sample on the internal standard

[0138]

[0139] After the blank sample was extracted, compared with the pure solution of QC M concentration, the deviation of the mean retention time of the chromatographic peak was within 0.1%. At the same time, the relative deviation of the ratio of the qualitative peak to the quantitative peak of the blank sample and the QC M sample was within 11.3%, which can prove that the chromatographic peak of the blank matrix is ​​produced by endogenous analytes, and the interference of other co-eluting substances in the blank matrix is ​​excluded (Table 4).

[0140] Table 4 Peak purity investigation

[0141]

[0142] 4.2 Linear range of standard curve

[0143] Prepare two samples of the standard curve, including 8 concentrations, to establish a standard curve. The concentration of the analyte in the biological sample (C) is used as the horizontal axis (X), and the peak area ratio of the analyte to the internal standard is used as the vertical axis (Y). The weighted least squares method (W = 1 / X 2) to perform regression calculation and obtain the linear regression equation, which is the standard curve. The concentration of each standard curve sample is calculated by the regression equation, but the blank and zero concentration samples are not included in the calculation. The deviation of the back-calculated concentration [(calculated value-theoretical value) / theoretical value×100%] should be within ±15% (LLOQ is ±20%). 75% of the samples should have at least 50% of each concentration meet the above requirements. Unqualified samples should be discarded and re-calculated.

[0144] The results showed that the serum concentration of testosterone-d3 was linear in the range of 10.0 to 10000 pg / mL, and the results met the method requirements (Table 5). The typical standard curve of testosterone-d3 in serum is shown in Figure 8 .

[0145] Table 5 Accuracy of each concentration of testosterone-d3 standard curve samples in SD rat serum (% Bias)

[0146]

[0147] 4.3 Precision and accuracy

[0148] Prepare 6 copies of LLOQ, low, medium and high concentration testosterone-d3 quality control samples (QC), calculate the concentration of QC samples with the standard curve of the batch, and use this to calculate the precision and accuracy of the method (prepare and measure at least 3 batches, and perform for at least two days). It is required that the intra-batch and inter-batch precision (RSD) of low, medium and high concentration QC samples should not exceed 15%, and LLOQ should not exceed 20%; the accuracy (Bias) of the intra-batch and inter-batch average values ​​of low, medium and high concentration QC samples should be within the range of ±15%, and LLOQ should be within the range of ±20%;

[0149] Prepare 6 copies of low, medium and high concentration testosterone quality control samples (QC) and 3 copies of blank control samples. Calculate the concentration of QC samples with the testosterone-d3 standard curve of the batch (minus the concentration of blank control samples), and use this to calculate the precision and accuracy of the method (prepare and measure at least 3 batches, and perform for at least two days). It is required that the intra-batch and inter-batch precision (RSD) of low, medium and high concentration QC samples should not exceed 15%, and the LLOQ should not exceed 20%; the accuracy (Bias) of the intra-batch and inter-batch averages of low, medium and high concentration QC samples should be within the range of ±15%, and the LLOQ should be within the range of ±20%; the accuracy (Bias) of QC samples should be within the range of ±15%, and the LLOQ should be within the range of ±20%.

[0150] The results showed that the LLOQ intra-batch precision (RSD) of testosterone-d3 was within 20%, and the mean accuracy (Bias) was within ±20%; the intra-batch precision (RSD) of low, medium and high concentration QC samples was within 15%, and the mean accuracy (Bias) was within ±15%; the LLOQ inter-batch precision (RSD) was within 20%, and the mean accuracy (Bias) was within ±20%; the inter-batch precision (RSD) of low, medium and high concentration QC samples was within 15%, and the mean accuracy (Bias) was within ±15%. All met the method requirements (Table 6).

[0151] Table 6 Accuracy and precision of testosterone-d3 in rat serum

[0152]

[0153]

[0154] The intra-batch precision (RSD) of the low, medium and high concentration QC samples of testosterone was within 15%, and the mean accuracy (Bias) was within ±15%; the inter-batch precision (RSD) of the low, medium and high concentration QC samples was within 15%, and the mean accuracy (Bias) was within ±15%. All met the method requirements (Appendix 6).

[0155] 5. Conclusion

[0156] The testosterone quantitative method in serum of the present invention has high specificity and accuracy and good reproducibility.

[0157] In summary, the present invention performs pretreatment on serum samples by a specific magnetic bead adsorption method, and under the cooperation of specific chromatographic mass spectrometry conditions, it is possible to accurately and quantitatively detect the content of testosterone in blood samples using only a trace amount of serum sample (100 μL), and the sensitivity is high and the detection limit is low, so that pg / ml level total testosterone in serum can also be measured, and the concentration range between the lowest concentration point and the highest concentration point of the standard curve can reach 1000 times, ensuring that when performing testosterone quantitative analysis, regardless of the concentration of the blood sample, it can be performed within the linear dynamic region of LC-MS / MS, thereby ensuring the reliability and accuracy of the analysis results.

Claims

1. A LC-MS / MS method for rapid quantitative analysis of testosterone concentration in serum, characterized in that: It includes the following steps: (1) Establishment of testosterone-d3 standard curve a. Preparation of working solution for series concentration standard curve: take testosterone-d3 standard substance, add acetonitrile to dissolve, and dilute the dissolved solution into series concentration by adding methanol solution; respectively take solutions of series concentration, add matrix, water and internal standard solution to mix, extract the mixed solution by magnetic bead adsorption method, and collect the eluate after extraction as the working solution for series concentration standard curve; The steps of the magnetic bead adsorption method are: The mixed solution is loaded onto the balanced magnetic bead solution, and then washed with an aqueous solution containing 0.05-0.15% formic acid and 30-40% methanol, and an aqueous solution containing 0.05-0.15% ammonia and 30-40% methanol, and then eluted with 70-90% methanol solution, and the eluate is collected; b. Pipette a series of concentration standard working solutions and inject them into the LC-MS / MS instrument to measure the peak area. Draw a testosterone-d3 standard curve with the ratio of the peak area of ​​testosterone-d3 to the internal standard as the ordinate and the concentration of the testosterone-d3 standard substance as the abscissa; The chromatographic conditions were as follows: Chromatographic column: Octyl bonded silica gel as filler; acetic acid-ammonium acetate aqueous solution as mobile phase A, formic acid methanol as mobile phase B, gradient elution program is: Mass spectrometry conditions: the ion source was electrospray ionization source; the mass spectrometry detection mode was multiple ion reaction monitoring; (2) Determination of testosterone content in the sample to be tested: c. Preparation of biological samples Take a serum sample to be tested, add water and internal standard solution to mix, extract the mixed solution according to the same magnetic bead adsorption method in step a, collect the eluate after extraction, and obtain a biological sample; d. Determination of biological samples Take a biological sample, inject it into the LC-MS / MS instrument for detection, and detect it under the same conditions as step b, and obtain the testosterone content in the serum sample to be tested according to the standard curve of step (1).

2. The LC-MS / MS method according to claim 1, characterized in that: The content determination process in step (2) also includes a quality control procedure, which specifically includes the following steps: Take the matrix, add water and internal standard solution in a volume ratio of 100:200:10 and mix well. At the same time, take testosterone-d3 standard substance and testosterone standard substance respectively, add acetonitrile to dissolve, dilute the dissolved solution with methanol solution to the same concentration, and then mix well with the matrix, water and internal standard solution in a volume ratio of 10:90:200:10; The mixed solution is extracted according to the same magnetic bead adsorption method as in step a, the eluate after extraction is collected, injected into the LC-MS / MS instrument, and the interference in the determination process is monitored; The concentration of the dissolving solution is 1.00 mg / mL, and the dissolving solution is diluted to a solution with a concentration of 0.1 to 100 ng / mL.

3. The LC-MS / MS method according to claim 1, characterized in that: The volume ratio of the solution of the series of concentrations in step a, the matrix, water and the internal standard solution is 10:90:200:10; the concentration of testosterone-d3 in the solution of the series of concentrations is 0.1-100 ng / mL.

4. The LC-MS / MS method according to any one of claims 1 to 3, characterized in that: The matrix is ​​rat blank serum.

5. The LC-MS / MS method according to claim 1, characterized in that: Step a) The steps of the magnetic bead adsorption method are: After the magnetic bead solution is mixed and balanced with the balance solution, the sample mixture is adsorbed for 50-70 seconds, and then an aqueous solution containing 0.1% formic acid and 35% methanol, and an aqueous solution containing 0.1% ammonia and 35% methanol are added in sequence for elution, and finally an 80% methanol aqueous solution is added for elution, and the eluate is collected; The volume ratio of the magnetic bead solution, the balance solution, the mixed solution, the aqueous solution containing 0.1% formic acid and 35% methanol, the aqueous solution containing 0.1% ammonia water and 35% methanol, and the 80% methanol solution is 200 μL: 500 μL: 310 μL: 500 μL: 500 μL: 160 μL; The magnetic bead solution is an aqueous solution containing 5 mg / mL hydrophobic magnetic polymer microspheres and 30% methanol; The balance solution is a 5% methanol aqueous solution; The mixing equilibrium time is 20 s, the adsorption temperature is 45° C., the time is 60 s, the elution time is 20 s, the elution temperature is 45° C., and the time is 60 s.

6. The LC-MS / MS method according to claim 1 or 3, characterized in that: The internal standard solution is acetonitrile containing testosterone-d5 standard substance, and the methanol solution is diluted to a concentration of 1.00ng / mL~10.0ng / mL solution.

7. The LC-MS / MS method according to claim 1, characterized in that: In the chromatographic conditions of step b, the chromatographic column is ACQUITY UPLC BEH C8 1.8 μm 2.1×50 mm Column, the injection volume is 5 μL, the column temperature is 40° C., and the flow rate is 0.6 ml / min.

8. The LC-MS / MS method according to claim 1, characterized in that: The mobile phase A is 0.1% acetic acid-2 mM ammonium acetate aqueous solution; the mobile phase B is 0.1% formic acid methanol.

9. The LC-MS / MS method according to claim 1, characterized in that: In the mass spectrometry conditions of step b, curtain gas pressure CUR: 35 psi; collision gas pressure CAD: 9.00 psi; ionization voltage IS: 5500 V; nebulization temperature TEM: 500° C.; nebulization gas pressure 1GS1: 55 psi; nebulization gas pressure 2GS2: 55 psi; scanning time: 4.0 min; declustering voltage DP: 100 V, entrance voltage EP: 10 V, collision voltage CXP 11 V.

10. The LC-MS / MS method according to claim 8, characterized in that: In the mass spectrometry conditions, the testosterone quantitative ion pair is m / z 289.2→m / z 97.1, and the collision energy CE is 27 eV; the testosterone qualitative ion pair is m / z 289.2→m / z 109.0, and the collision energy CE is 33 eV; the testosterone-d3 quantitative ion pair is m / z 292.2→m / z 97.1, and the collision energy CE is 25 eV; the testosterone-d5 quantitative ion pair is m / z 294.2→m / z 100.0, and the collision energy CE is 30 eV.

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