阅读说明：本技术 肝微粒体孵育体系中睾酮及6β-羟基睾酮检测平台的建立方法和使用方法 (Establishment method and use method of testosterone and 6 beta-hydroxytestosterone detection platform in liver microsome incubation system ) 是由 李明 盘建红 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种肝微粒体孵育体系中睾酮及6β-羟基睾酮检测平台的建立方法,包括如下步骤：采用甲醇稀释混合储备液,得到标准曲线工作液,任意一个浓度的标准曲线工作液中睾酮和6β-羟基睾酮的浓度相同。采用肝微粒体孵育体系稀释标准曲线工作液,得到标准曲线样品。取100ng/mL的标准曲线样品,加入含卡马西平的甲醇沉淀剂,振荡、离心后,取上清加入超纯水,采用LC-MS/MS进样分析。得出睾酮最优质谱条件和6β-羟基睾酮最优质谱条件。设置睾酮最优质谱条件以及6β-羟基睾酮最优质谱条件,将标准曲线样品采用LC-MS/MS进样分析,得到睾酮线性回归方程和6β-羟基睾酮线性回归方程。本发明方法缩短了分析时间,提高了检测效率,确保试验结果的准确性,并且符合方法学验证要求。(The invention discloses a method for establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system, which comprises the following steps: and diluting the mixed stock solution by using methanol to obtain a standard curve working solution, wherein the concentrations of testosterone and 6 beta-hydroxytestosterone in the standard curve working solution with any concentration are the same. And (4) diluting the standard curve working solution by adopting a liver microsome incubation system to obtain a standard curve sample. And (3) adding a methanol precipitator containing carbamazepine into a 100ng/mL standard curve sample, oscillating and centrifuging, taking the supernatant, adding ultrapure water, and performing sample injection analysis by adopting LC-MS/MS. Obtaining the optimal mass spectrum condition of testosterone and the optimal mass spectrum condition of 6 beta-hydroxytestosterone. And setting optimal mass spectrum conditions of testosterone and 6 beta-hydroxytestosterone, and analyzing the sample of the standard curve by adopting LC-MS/MS sample injection to obtain a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation. The method shortens the analysis time, improves the detection efficiency, ensures the accuracy of the test result, and meets the requirement of methodology verification.)

1. A method for establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system is characterized by comprising the following steps:

step one, determination of optimal mass spectrum conditions

Establishing a liver microsome incubation system containing human liver microsomes, NADPH, magnesium chloride and PBS buffer solution;

preparing a mixed stock solution, wherein the concentrations of testosterone and 6 beta-hydroxytestosterone in the mixed stock solution are the same;

diluting the mixed stock solution by using methanol to obtain standard curve working solutions with the testosterone series concentration of 5-5000 ng/mL, wherein the concentrations of testosterone and 6 beta-hydroxytestosterone in any standard curve working solution with any concentration are the same;

diluting the standard curve working solution by adopting a liver microsome incubation system to obtain standard curve samples with the testosterone series concentration of 0.5-500 ng/mL;

taking a 100ng/mL standard curve sample, adding a methanol precipitator containing carbamazepine, oscillating, centrifuging, taking the supernatant, adding ultrapure water, and performing sample injection analysis by adopting LC-MS/MS;

wherein the first mass spectrometric conditions for testosterone are set separately as: ion pair m/z 289.4 → m/z 109.1, cone aperture voltage 90V, collision voltage 40V; the second mass spectral conditions were: ion pair m/z 289.4 → m/z 97.1, cone hole voltage 90V, collision voltage 45V; the third mass spectral conditions were: ion pair m/z 289.4 → m/z 88.0, cone aperture voltage 90V, collision voltage 55V; comparing the detection results to obtain the optimal mass spectrum condition of testosterone;

wherein, the fourth mass spectrum condition is respectively set for the 6 beta-hydroxytestosterone as follows: ion pair m/z 305.1 → m/z 287.3, cone hole voltage 50V, collision voltage 20V; the fifth mass spectral conditions were: ion pair m/z 305.1 → m/z 269.3, cone aperture voltage 50V, collision voltage 22V; the sixth mass spectral conditions were: ion pair m/z 305.1 → m/z 227.2, cone hole voltage 50V and collision voltage 30V; comparing the detection results to obtain the optimal mass spectrum condition of the 6 beta-hydroxytestosterone;

step two, establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system

And setting optimal mass spectrum conditions of testosterone and 6 beta-hydroxytestosterone, and analyzing a standard curve sample with the testosterone series concentration of 0.5-500 ng/mL by adopting LC-MS/MS sample injection to obtain a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

2. The method for establishing a platform for detecting testosterone and 6 β -hydroxytestosterone in a liver microsome incubation system according to claim 1, wherein the optimal mass spectrometry condition for testosterone is a first mass spectrometry condition.

3. The method for establishing a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system according to claim 1, wherein the optimal mass spectrometry condition of 6 beta-hydroxytestosterone is a fifth mass spectrometry condition.

4. The method for establishing a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system according to claim 1, wherein the LC-MS/MS analysis comprises the following liquid chromatography conditions:

flow rate: 0.5 mL/min; mobile phase A: 0.1% aqueous formic acid containing 2mM ammonium acetate; mobile phase B: acetonitrile; elution time length: 1.5 min; and (3) an elution mode: gradient elution with a total flow rate of 0.5 mL/min; column temperature: 40 ℃; autosampler temperature: 4 ℃; sample introduction volume: 5 mu L of the solution; retention time: testosterone for 1.1min, 6 beta-hydroxytestosterone for 0.9min, and carbamazepine for 1.2 min.

5. The method for establishing a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system according to claim 4, wherein the gradient elution procedure in the LC-MS/MS analysis comprises:

0.2min, the volume percentage of the mobile phase A is 90 percent, and the volume percentage of the mobile phase B is 10 percent;

0.5min, the volume percentage of the mobile phase A is 10 percent, and the volume percentage of the mobile phase B is 90 percent;

1.0min, the volume percentage of the mobile phase A is 10 percent, and the volume percentage of the mobile phase B is 90 percent;

1.2min, the volume percentage of the mobile phase A is 90 percent, and the volume percentage of the mobile phase B is 10 percent;

for 1.5min, the volume percentage of the mobile phase A is 90%, and the volume percentage of the mobile phase B is 10%.

6. The method for establishing a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system according to claim 1, wherein the mass spectrometry conditions in the LC-MS/MS analysis are as follows:

an ion source: an electrospray ion source; ionization mode: a positive ion mode; the scanning mode is as follows: multi-reaction detection scanning; collision gas: 9 psi; air curtain air: 20 psi; resolution Q1/Q3: Unit/Unit; spraying voltage: 5500 v; ionization temperature: 500 ℃; analysis time: 1.5 min.

7. The method for establishing a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system according to claim 6, wherein in the LC-MS/MS analysis, the mass spectrum conditions of testosterone are as follows: q1: 289.4 Da; q3: 109.1 Da; time: 100 ms; de-clustering voltage: 90V; collision voltage: 40V; collision cell emission voltage: 15V; injection voltage: 10V;

the mass spectrum conditions of the 6 beta-hydroxytestosterone are as follows: q1: 305.1 Da; q3: 269.3 Da; time: 100 ms; de-clustering voltage: 50V; collision voltage: 22V; collision cell emission voltage: 15V; injection voltage: 10V;

the mass spectrum conditions of the carbamazepine are as follows: q1: 237.1 Da; q3: 194.1 Da; time: 100 ms; de-clustering voltage: 60V; collision voltage: 24V; collision cell emission voltage: 15V; injection voltage: 10V.

8. A method for using a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system, which adopts the detection platform established by the establishment method of the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system according to any one of claims 1 to 7, and is characterized by comprising the following steps:

incubating testosterone in a liver microsome incubation system to obtain an incubation substance;

taking 200 mu L of the incubated substance, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking up, and performing LC-MS/MS sample injection analysis; wherein the concentration of the methanol precipitator, namely carbamazepine, is 10 ng/mL;

and calculating the concentrations of the testosterone and the specific product 6 beta-hydroxytestosterone thereof in the culture according to a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

Technical Field

The invention relates to a method for establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system and a use method thereof.

Background

Cytochrome P closely related to drug metabolism₄₅₀Enzyme (CYP)₄₅₀) The liver cancer treatment method mainly comprises CYP 1-4, wherein the CYP3A subfamily is the most important, and CYP3A4 is the main component in adult livers.

CYP3A4 has more substrates, and about 150 drugs in 38 classes are counted as the substrates of the drugs, which account for about 50% of all the drugs, so that the accurate evaluation of the enzymatic activity of CYP3A4 is very important for drug interaction research and clinical reasonable drug administration.

At present, testosterone is mostly adopted at home and abroad as a probe substrate of CYP3A4, and the activity of the enzyme is evaluated by measuring the reduction amount of the testosterone or the generation amount of a metabolite 6 beta-hydroxytestosterone in the in vitro metabolic reaction process of liver microsomes.

The traditional test method is poor in accuracy, and the activity of CYP3A4 is difficult to accurately evaluate. Therefore, it is necessary to establish a platform for detecting testosterone and 6 beta-hydroxytestosterone in a liver microsome incubation system.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a method for establishing and using a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system, which shortens the analysis time, improves the detection efficiency, ensures the accuracy of test results and meets the requirement of methodology verification.

The invention discloses a method for establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system, which comprises the following steps:

step one, determination of optimal mass spectrum conditions

Establishing a liver microsome incubation system containing human liver microsomes, NADPH, magnesium chloride and PBS buffer solution;

preparing a mixed stock solution, wherein the concentrations of testosterone and 6 beta-hydroxytestosterone in the mixed stock solution are the same;

diluting the mixed stock solution by using methanol to obtain standard curve working solutions with the testosterone series concentration of 5-5000 ng/mL, wherein the concentrations of testosterone and 6 beta-hydroxytestosterone in any standard curve working solution with any concentration are the same;

diluting the standard curve working solution by adopting a liver microsome incubation system to obtain standard curve samples with the testosterone series concentration of 0.5-500 ng/mL;

taking a 100ng/mL standard curve sample, adding a methanol precipitator containing carbamazepine, oscillating, centrifuging, taking the supernatant, adding ultrapure water, and performing sample injection analysis by adopting LC-MS/MS;

wherein the first mass spectrometric conditions for testosterone are set separately as: ion pair m/z 289.4 → m/z 109.1, cone aperture voltage 90V, collision voltage 40V; the second mass spectral conditions were: ion pair m/z 289.4 → m/z 97.1, cone hole voltage 90V, collision voltage 45V; the third mass spectral conditions were: ion pair m/z 289.4 → m/z 88.0, cone aperture voltage 90V, collision voltage 55V; comparing the detection results to obtain the optimal mass spectrum condition of testosterone;

wherein, the fourth mass spectrum condition is respectively set for the 6 beta-hydroxytestosterone as follows: ion pair m/z 305.1 → m/z 287.3, cone hole voltage 50V, collision voltage 20V; the fifth mass spectral conditions were: ion pair m/z 305.1 → m/z 269.3, cone aperture voltage 50V, collision voltage 22V; the sixth mass spectral conditions were: ion pair m/z 305.1 → m/z 227.2, cone hole voltage 50V and collision voltage 30V; comparing the detection results to obtain the optimal mass spectrum condition of the 6 beta-hydroxytestosterone;

step two, establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system

And setting optimal mass spectrum conditions of testosterone and 6 beta-hydroxytestosterone, and analyzing a standard curve sample with the testosterone series concentration of 0.5-500 ng/mL by adopting LC-MS/MS sample injection to obtain a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

Preferably, the testosterone optimum mass spectrometry conditions are first mass spectrometry conditions.

Preferably, the optimal mass spectrometry condition for 6 β -hydroxytestosterone is a fifth mass spectrometry condition.

Preferably, the liquid chromatography conditions in the LC-MS/MS analysis are as follows:

flow rate: 0.5 mL/min; mobile phase A: 0.1% aqueous formic acid containing 2mM ammonium acetate; mobile phase B: acetonitrile; elution time length: 1.5 min; and (3) an elution mode: gradient elution with a total flow rate of 0.5 mL/min; column temperature: 40 ℃; autosampler temperature: 4 ℃; sample introduction volume: 5 mu L of the solution; retention time: testosterone for 1.1min, 6 beta-hydroxytestosterone for 0.9min, and carbamazepine for 1.2 min.

Further preferably, the gradient elution procedure in the LC-MS/MS analysis is:

0.2min, the volume percentage of the mobile phase A is 90 percent, and the volume percentage of the mobile phase B is 10 percent;

0.5min, the volume percentage of the mobile phase A is 10 percent, and the volume percentage of the mobile phase B is 90 percent;

1.0min, the volume percentage of the mobile phase A is 10 percent, and the volume percentage of the mobile phase B is 90 percent;

1.2min, the volume percentage of the mobile phase A is 90 percent, and the volume percentage of the mobile phase B is 10 percent;

for 1.5min, the volume percentage of the mobile phase A is 90%, and the volume percentage of the mobile phase B is 10%.

Preferably, the mass spectrum conditions in the LC-MS/MS analysis are as follows:

an ion source: an electrospray ion source; ionization mode: a positive ion mode; the scanning mode is as follows: multi-reaction detection scanning; collision gas: 9 psi; air curtain air: 20 psi; resolution Q1/Q3: Unit/Unit; spraying voltage: 5500 v; ionization temperature: 500 ℃; analysis time: 1.5 min.

Further preferably, in the LC-MS/MS analysis, the mass spectrometric conditions of testosterone are: q1: 289.4 Da; q3: 109.1 Da; time: 100 ms; de-clustering voltage: 90V; collision voltage: 40V; collision cell emission voltage: 15V; injection voltage: 10V;

the mass spectrum conditions of the 6 beta-hydroxytestosterone are as follows: q1: 305.1 Da; q3: 269.3 Da; time: 100 ms; de-clustering voltage: 50V; collision voltage: 22V; collision cell emission voltage: 15V; injection voltage: 10V;

the mass spectrum conditions of the carbamazepine are as follows: q1: 237.1 Da; q3: 194.1 Da; time: 100 ms; de-clustering voltage: 60V; collision voltage: 24V; collision cell emission voltage: 15V; injection voltage: 10V.

The invention also provides a use method of the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system, the detection platform established by the establishment method of the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system comprises the following steps:

incubating testosterone in a liver microsome incubation system to obtain an incubation substance;

taking 200 mu L of the incubated substance, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking up, and performing LC-MS/MS sample injection analysis; wherein the concentration of the methanol precipitator, namely carbamazepine, is 10 ng/mL;

and calculating the concentrations of the testosterone and the specific product 6 beta-hydroxytestosterone thereof in the culture according to a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

The invention has the following beneficial effects:

the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system established by the invention can be used for simultaneously detecting testosterone prototypes and metabolites, so that the analysis time is shortened and the detection efficiency is improved aiming at some tests needing simultaneous detection and analysis. Aiming at some tests which do not need to be detected and analyzed simultaneously, joint detection is equivalently added, and the accuracy of test results is ensured.

The system methodology verification is carried out on the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system established by the invention, so that the accuracy of the detection result is ensured.

The testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system established by the invention has the advantages of small matrix effect, high accuracy and precision in batch and batch, high stability, accordance with the requirement of methodology verification and wide application prospect.

In order to make the aforementioned and other objects, features and advantages of the invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a preferred result of parent ion and cone voltage in testosterone LC-MS/MS in an embodiment of the present invention;

FIG. 2 is a preferred result of parent ion and cone voltage in 6 β -hydroxytestosterone LC-MS/MS in the examples of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment provides a method for establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system, and the blank matrix in the embodiment is a liver microsome incubation system containing human liver microsomes, NADPH, magnesium chloride and PBS buffer solution. In this example, the concentration of carbamazepine in the methanol precipitant is 10ng/mL, and carbamazepine is the internal standard substance in this example.

The method for establishing the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system comprises the following steps:

step one, determination of optimal mass spectrum conditions

(1) Establishing a liver microsome incubation system containing human liver microsomes, NADPH, magnesium chloride and PBS buffer.

(2) Preparing mixed stock solution

Preparing a testosterone stock solution with testosterone concentration of 1mg/mL by adopting methanol and a testosterone standard substance;

preparing 6 beta-hydroxytestosterone stock solution with the concentration of 6 beta-hydroxytestosterone of 1mg/mL by adopting methanol and a 6 beta-hydroxytestosterone standard substance;

and mixing the testosterone stock solution and the 6 beta-hydroxytestosterone stock solution according to the volume ratio of 1:1 to obtain a mixed stock solution with the concentrations of testosterone and 6 beta-hydroxytestosterone both being 0.5 mg/mL.

(3) Preparing a standard curve sample

And diluting the mixed stock solution by adopting methanol to obtain standard curve working solutions with testosterone series concentrations of 5, 10, 30, 100, 300, 1000, 3000 and 5000 ng/mL. The concentrations of testosterone and 6 beta-hydroxytestosterone in the working solution of any one concentration standard curve are the same.

And mixing the standard curve working solution and the liver microsome incubation system according to the volume ratio of 1:9 to obtain standard curve samples with the testosterone series concentrations of 0.5, 1, 3, 10, 30, 100, 300 and 500 ng/mL. The concentration of testosterone and 6 β -hydroxytestosterone was the same in any concentration of the standard curve sample.

(4) Configuring quality control sample

And diluting the mixed stock solution by adopting methanol to obtain testosterone series quality control working solutions with the concentrations of 5, 150, 2500 and 4000 ng/mL. The concentration of testosterone and 6 beta-hydroxytestosterone in any concentration of quality control working solution is the same.

And mixing the quality control working solution and the liver microsome incubation system according to the volume ratio of 1:9, and shaking up to obtain quality control samples with testosterone series concentrations of 0.5, 15, 250 and 400 ng/mL. The concentration of testosterone and 6 beta-hydroxytestosterone in any concentration of quality control sample is the same.

(5) Analysis of sample introduction

And (3) adding a methanol precipitator containing carbamazepine into a 100ng/mL standard curve sample, oscillating and centrifuging, taking the supernatant, adding ultrapure water, and performing sample injection analysis by adopting LC-MS/MS.

And respectively setting a first mass spectrum condition, a second mass spectrum condition and a third mass spectrum condition for the testosterone analysis sample, and comparing detection results to obtain an optimal mass spectrum condition.

Wherein the first mass spectrometry conditions are: ion pair m/z 289.4 → m/z 109.1, cone aperture voltage 90V, collision voltage 40V, the result shows that the intensity is 2.55X 10⁵。

The second mass spectral conditions were: ion pair m/z 289.4 → m/z 97.1, taper hole voltage 90V, impact voltage 45V, the result shows that the intensity is 2.40X 10⁵。

The third mass spectral conditions were: ion pair m/z 289.4 → m/z 88.0, cone aperture voltage 90V, collision voltage 55V, the result shows that the intensity is 1.35X 10⁵。

FIG. 1 shows the preferred results of parent ion and cone-hole voltages, with the first mass spectral condition selected, taking into account the spectra and intensity results.

And respectively setting a fourth mass spectrum condition, a fifth mass spectrum condition and a sixth mass spectrum condition for the 6 beta-hydroxytestosterone analysis sample, and comparing detection results to obtain an optimal mass spectrum condition.

Wherein the fourth mass spectrometry conditions are: ion pair m/z 305.1 → m/z 287.3, cone hole voltage 50V, collision voltage 20V, and the result shows that the intensity is 1.32 × 10⁵。

The fifth mass spectral conditions were: ion pair m/z 305.1 → m/z 269.3, cone aperture voltage 50V, collision voltage 22V, and the result shows that the intensity is 1.65X 10⁵。

The sixth mass spectral conditions were: ion pair m/z 305.1 → m/z 227.2, cone hole voltage 50V, collision voltage 30V, the result shows that the intensity is 8.50X 10⁴。

FIG. 2 shows the preferred results of parent ion and cone voltage, with the spectrum and intensity taken into account, and the fifth mass spectral condition selected.

Step two, establishing a testosterone and 6 beta-hydroxytestosterone detection platform in a liver microsome incubation system

And setting the mass spectrum condition of testosterone as a first mass spectrum condition and the mass spectrum condition of 6 beta-hydroxytestosterone as a fifth mass spectrum condition, and analyzing the sample of the standard curve by adopting LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) to obtain a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

In the first and second steps of the present embodiment, the liquid chromatography conditions in the LC-MS/MS analysis are shown in tables 1 and 2:

TABLE 1 liquid chromatography conditions

TABLE 2 gradient elution procedure

Mass Spectrometry conditions for LC-MS/MS are shown in Table 3 and Table 4.

Table 3 mass spectrometric conditions 1

Table 4 mass spectrometric conditions 2

Step three, in the establishment of the testosterone detection platform, the methodological verification of the LC-MS/MS test

(1) Intra-and inter-batch precision/accuracy

In the quality control sample of the embodiment, the concentration of testosterone comprises: the lower limit of quantitation (LLOQ) is 0.5ng/mL, the low concentration (QC3) is 15ng/mL, the medium concentration (QC2) is 250ng/mL, and the high concentration (QC1) is 400 ng/mL. Each concentration of the quality control sample also contained 6 β -hydroxytestosterone, and the 6 β -hydroxytestosterone concentration and testosterone concentration were the same, for example, the LLOQ sample contained 0.5ng/mL testosterone and 0.5ng/mL6 β -hydroxytestosterone.

Taking 200 mu L of quality control samples of each concentration, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant to a 96-well plate, adding 100 mu L of ultrapure water, shaking uniformly, carrying out LC-MS/MS sample injection analysis, setting 6 groups of parallel concentrations, continuously measuring 3 batches (the internal precision/accuracy-1, the internal precision/accuracy-2 and the internal precision/accuracy-3) to calculate the concentration of the samples according to a batch standard curve, and analyzing the batch precision/accuracy and the internal precision/accuracy according to the test results. When the samples are measured, each quality control sample is analyzed once, and results of testosterone and 6 beta-hydroxytestosterone are detected simultaneously. The testosterone dependent results are shown in tables 5-8, and the 6 β -hydroxytestosterone dependent results are shown in tables 9-12.

For the lower limit of quantitation, the accuracy should be between 80-120% with a coefficient of variation of 20% or less. For the low, medium and high concentration groups, the accuracy should be between 85-115% with a coefficient of variation of 15% or less. As can be seen from the test and analysis results, the method of the present embodiment can meet the requirements.

TABLE 5 Testosterone in-batch precision/accuracy-1

TABLE 6 Testosterone in-batch precision/accuracy-2

TABLE 7 Testosterone in-batch accuracy/precision-3

TABLE 8 Testosterone Interbatch precision/accuracy

TABLE 96 beta-hydroxytestosterone in-batch precision & accuracy-1

TABLE 106 beta-hydroxytestosterone in-batch precision & accuracy-2

TABLE 116 beta-hydroxytestosterone in-batch precision & accuracy-3

TABLE 126 beta-hydroxytestosterone inter-batch precision & accuracy

(2) Standard curve validation

According to the method in the step one, standard curve samples with testosterone series concentrations of 0.5, 1, 3, 10, 30, 100, 300 and 500ng/mL are prepared, and the concentration of 6 beta-hydroxytestosterone and the concentration of testosterone in each concentration standard curve sample are the same.

And (3) taking 200 mu L of a standard curve sample of each concentration, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant to a 96-well plate, adding 100 mu L of ultrapure water, shaking uniformly, and carrying out LC-MS/MS sample injection analysis.

Each concentration was run in parallel with 6 groups, each sample was analyzed only once and both components were tested simultaneously, the results of the testosterone tests and analyses are shown in table 13, and the results of the 6 β -hydroxytestosterone tests and analyses are shown in table 14.

The results show that the linear relation between the linear regression equation of testosterone and the linear regression equation of 6 beta-hydroxytestosterone in the detection method of the embodiment is good.

TABLE 13 Testosterone Standard Curve validation

TABLE 146 beta-hydroxytestosterone Standard Curve validation

(3) Matrix Effect and extraction recovery

And (3) extracting a sample: and (3) taking 200 mu L of quality control samples with testosterone concentrations of 15ng/mL (QC3), 250ng/mL (QC2) and 400ng/mL (QC1) respectively, adding 200 mu L of methanol precipitator containing carbamazepine into each concentration of quality control samples, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking up, and analyzing by LC-MS/MS sample injection. And 6 groups of concentration are paralleled to obtain peak area data of the extract sample, the concentration of 6 beta-hydroxytestosterone in the quality control sample of each concentration is the same as that of testosterone, each sample is analyzed once only, two components are detected simultaneously, the test result of the testosterone extract sample is shown in a table 15, and the test result of the 6 beta-hydroxytestosterone extract sample is shown in a table 16.

Adding a sample after extraction: adding 200 μ L methanol precipitant containing carbamazepine into 180 μ L blank matrix, mixing with 20 μ L quality control working solution to obtain extracts with testosterone concentrations of 15ng/mL (QC3), 250ng/mL (QC2) and 400ng/mL (QC1), adding the samples, oscillating, centrifuging, collecting supernatant 100 μ L, transferring to 96-well plate, adding 100 mu L of ultrapure water, shaking up, performing LC-MS/MS sample injection analysis, wherein each concentration is 6 groups in parallel to obtain peak area data of the sample added after extraction, the concentration of 6 beta-hydroxytestosterone in the quality control working solution of each concentration is the same as that of testosterone, each sample is only subjected to one-time analysis, the two components were tested simultaneously, the results of the addition of the sample after testosterone extraction are shown in table 17, and the results of the addition of the sample after 6 β -hydroxytestosterone extraction are shown in table 18.

Matrix-free samples: adding 200 mu L of methanol precipitant containing carbamazepine into 180 mu L of ultrapure water, mixing with 20 mu L of quality control working solution to obtain matrix-free samples with testosterone concentrations of 15ng/mL (QC3) and 400ng/mL (QC1) respectively, oscillating, centrifuging, taking 100 mu L of supernatant, transferring to a 96-well plate, adding 100 mu L of ultrapure water, shaking, performing LC-MS/MS sample injection analysis, wherein each concentration is 6 groups in parallel to obtain peak area data of the matrix-free samples, the concentration of 6 beta-hydroxytestosterone in the quality control working solution of each concentration is the same as that of testosterone, each sample is subjected to one-time analysis only, and two components are detected simultaneously, and the test result of the testosterone matrix-free samples is shown in Table 19, and the test result of the 6 beta-hydroxytestosterone matrix-free samples is shown in Table 20. .

The analysis of the test results of the extract samples, the added samples after extraction and the non-matrix samples resulted in the data related to the matrix effect and the recovery rate, the recovery rate of testosterone is shown in table 21, the recovery rate of 6 beta-hydroxytestosterone is shown in table 22, the matrix effect of testosterone is shown in table 23, and the matrix effect of 6 beta-hydroxytestosterone is shown in table 24. The results show that the test method of this example has low matrix effect and high extraction recovery.

TABLE 15 Peak area data for Testosterone extract samples

TABLE 166 Peak area data for beta-hydroxytestosterone extract samples

TABLE 17 Peak area data for post-testosterone addition samples

TABLE 186 Peak area data of post-extraction addition samples of beta-hydroxytestosterone

TABLE 19 Peak area data for testosterone no matrix samples

TABLE 206 Peak area data for matrix free samples of beta-hydroxytestosterone

TABLE 21 recovery of testosterone

TABLE 226 beta-hydroxytestosterone recovery

TABLE 23 Testosterone matrix Effect

TABLE 246 beta-hydroxytestosterone matrix Effect

(4) Stability of

200 mu L of quality control samples with testosterone concentration of 15ng/mL at low concentration (QC3) and 400ng/mL at high concentration (QC1) are taken, and the concentration of testosterone and 6 beta-hydroxytestosterone in each concentration of quality control samples is the same. Adding 200 mu L of methanol precipitator containing carbamazepine into quality control samples with each concentration, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking uniformly, placing the mixture into a sample injector, placing the mixture for 24h, then carrying out sample injection analysis, wherein 3 groups of each concentration are parallel, each sample is only subjected to one-time analysis, two components are simultaneously detected, and the stability test result of the testosterone sample injector is shown in a table 25, and the stability test result of the 6 beta-hydroxytestosterone sample injector is shown in a table 26.

Taking 200 mu L of quality control samples with testosterone concentrations of 15ng/mL (QC3) and 400ng/mL (QC1) respectively, placing the quality control samples at room temperature for 6h, then adding 200 mu L of methanol precipitator containing carbamazepine into each concentration of quality control samples, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking up, carrying out sample injection analysis, enabling 3 groups of each concentration to be parallel, carrying out one-time analysis on each sample, and simultaneously detecting two components, wherein the room temperature stability test result of testosterone is shown in table 27, and the room temperature stability test result 28 of 6 beta-hydroxytestosterone.

As can be seen from the test data, the method of the present embodiment has high stability.

Table 25 testosterone sampler stability test

TABLE 266 beta-hydroxytestosterone injector stability test

TABLE 27 Testosterone Room temperature stability test

TABLE 286 beta-hydroxytestosterone Room temperature stability test

(5) Residue is remained

Taking 200 mu L of a standard curve sample with the high concentration of 500ng/mL, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant to a 96-well plate, adding 100 mu L of ultrapure water, and shaking up.

Blank samples without testosterone and 6 β -hydroxytestosterone were prepared according to the standard curve sample method.

(6) Testosterone standard curve samples and blank samples were injected alternately, and 6 sets of experiments were run in parallel. Each sample is analyzed once, two components are detected simultaneously, the peak area data of testosterone is shown in a table 29, the peak area data of testosterone residue is shown in a table 30, the peak area data of 6 beta-hydroxytestosterone is shown in a table 31, and the peak area data of 6 beta-hydroxytestosterone is shown in a table 32.

As can be seen from the test results, the method is residue-free.

TABLE 29 Testosterone Peak area data

TABLE 30 Testosterone residual test

TABLE 316 beta-hydroxytestosterone Peak area data

TABLE 326 beta-hydroxytestosterone residual test

The methodological verification test shows that the testosterone and 6 beta-hydroxytestosterone detection platform in the liver microsome incubation system established by the invention has the advantages of small matrix effect, high accuracy and precision in batch and batch, high stability, accordance with the methodological verification requirement and wide application prospect.

Step four, using testosterone and 6 beta-hydroxytestosterone detection platform in liver microsome incubation system

And (3) incubating testosterone in a liver microsome incubation system to obtain an incubation substance. Taking 200 mu L of the incubated substance, adding 200 mu L of methanol precipitator containing carbamazepine, oscillating, centrifuging, taking 100 mu L of supernatant, transferring the supernatant into a 96-well plate, adding 100 mu L of ultrapure water, shaking up, and carrying out sample injection analysis by 5 mu L of LC-MS/MS; wherein the concentration of the methanol precipitator, namely carbamazepine, is 10 ng/mL;

and calculating the concentrations of the testosterone and the specific product 6 beta-hydroxytestosterone thereof in the culture according to a testosterone linear regression equation and a 6 beta-hydroxytestosterone linear regression equation.

The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.