Liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in CYP inhibition test

Abstract

The invention discloses a liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test, which comprises the following steps of: selecting phenacetin, bupropion, amodiaquine, diclofenac, S-mephentoin, methaphan and midazolam as corresponding substrates of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A respectively; the activity of the CYP450 enzyme in the human liver microsome is reflected by simultaneously detecting the generation amounts of seven metabolites, namely acetaminophen, hydroxybupropion, N-deethylated amodiaquine, 4-hydroxydiclofenac, 4-hydroxymephentoin, dextrorphan and 1-hydroxymidazolam. According to the detection method provided by the invention, the problems of mutual interference and matrix effect among various metabolites are avoided, the metabolites can be rapidly and accurately determined, meanwhile, the detection flux can be improved, the detection efficiency can be improved, the actual requirements of drug research, development and screening can be met, and the scientificity and reliability of a drug evaluation result can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical analysis, and in particular to a liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test. Background Art

[0002] The development of innovative drugs is a high-investment and time-consuming process, which requires a large number of in vitro and in vivo pharmacokinetic studies. One of the key links is the need to evaluate the possibility of drug-drug interactions (DDI) when combined with other drugs. DDI is mainly related to the inhibition of cytochrome P450 enzyme (CYP) activity.

[0003] In order to achieve the purpose of treatment in clinical practice, combination therapy is often used, that is, two or more drugs are taken simultaneously or successively. The increase in the number of medications increases the probability of drug interactions, which may enhance or reduce the efficacy of the drug, or even produce adverse reactions. In addition, some foods may also have some adverse reactions with the drug during medication. In order to predict and reduce these adverse reactions in advance, in vitro drug-drug interaction (DDI) assessments are usually performed in the early stages of drug development. Among them, one of the key mechanisms leading to DDI is through the inhibition of the activity of cytochrome P450 enzymes (CYP) in the liver. When evaluating CYP inhibition for candidate drugs, the FDA recommends CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A as the main human liver CYP subtypes for evaluation. When the candidate metabolizing enzyme has been determined in vitro, the main metabolic pathway (≥25% of the total clearance) usually requires additional clinical characterization to determine and quantify the risk of candidate drugs interacting as DDI-affected drugs. Therefore, the evaluation of CYP enzyme activities is crucial in drug development, clinical or toxicology fields.

[0004] At present, single enzyme incubation method and cocktail method (also known as N-in-one or box incubation) can be used for enzyme activity evaluation. Single enzyme incubation method refers to evaluating the activity of only one specific enzyme in the experiment, while the cocktail method can evaluate the activity of multiple enzymes in the same experiment, that is, a variety of different enzyme substrates are given in one experiment, and the activity data of multiple enzymes can be obtained simultaneously by detecting the amount of metabolites. The high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) method is often used for late-stage detection of enzyme activity evaluation due to its high sensitivity and good selectivity. In the early stages of new drug development, a large number of samples will be involved in the testing, and there are higher requirements for throughput and completion cycle, so the cocktail method will be used first.

[0005] There are still many challenges when the cocktail method is combined with the LC-MS / MS method to evaluate CYP enzyme activity: the substrates metabolized by multiple different enzymes are incubated in a box, and the components in the sample system are relatively complex. It is necessary to examine the complex interference between multiple incubation substrates and multiple metabolites, as well as matrix effects and other issues. Furthermore, the currently commonly used cocktail method is a five-substrate box incubation (5-in-1), and the seven-substrate box incubation (7-in-1) is less used. In the prior art, the systematically verified 7-in-1 LC-MS / MS method has a long analysis time and cannot meet the high-throughput requirements in the early drug screening process. In addition, during the analysis, a wide variety of unknown candidate compounds may also interfere with the target analyte, and a single conventional internal standard cannot track this interference, so additional evaluation is required.

[0006] In summary, there is an urgent need in this field for a liquid chromatography-mass spectrometry detection method for simultaneously determining the metabolites of seven key probe substrates in CYP inhibition tests, avoiding mutual interference and matrix effect problems among multiple metabolites, and being able to quickly and accurately determine metabolites while increasing detection throughput and efficiency, thereby meeting the actual needs of drug development and screening and improving the scientificity and reliability of drug evaluation results. Summary of the invention

[0007] In order to solve the above technical problems, the present application selects phenacetin, bupropion, amodiaquine, diclofenac, S-mephenytoin, dextrorphan and midazolam as the corresponding substrates of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, and simultaneously detects the production amounts of 7 metabolites, namely acetaminophen, hydroxybupropion, N-desethylamodiaquine, 4-hydroxydiclofenac, 4-hydroxymephenytoin, dextrorphan and 1-hydroxymidazolam, to reflect the activity of CYP450 enzymes in human liver microsomes.

[0008] The present invention provides a liquid chromatography-mass spectrometry detection method for simultaneously determining metabolites of seven key probe substrates in a CYP inhibition test, characterized in that the probe substrates and metabolites of the CYP subtype to be evaluated in the CYP inhibition test are determined, and then the concentrations of all metabolites are simultaneously detected using a liquid chromatography-tandem mass spectrometry detection method, wherein the CYP subtype to be evaluated is CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, wherein:

[0009] The probe substrate of CYP1A2 is phenacetin, and the metabolite is acetaminophen;

[0010] The probe substrate of CYP2B6 is bupropion, and the metabolite is hydroxybupropion;

[0011] The probe substrate for CYP2C8 is amodiaquine, and the metabolite is N-desethylamodiaquine;

[0012] The probe substrate of CYP2C9 is diclofenac, and the metabolite is 4-hydroxydiclofenac;

[0013] The probe substrate for CYP2C19 is S-mephenytoin, and the metabolite is 4-hydroxymephenytoin;

[0014] The probe substrate of CYP2D6 is dextrorphan, and the metabolite is dextrorphan;

[0015] The probe substrate of CYP3A is midazolam and its metabolite is 1-hydroxymidazolam.

[0016] Specifically, the liquid chromatography tandem mass spectrometry detection is an internal standard method, and the internal standard substances are acetaminophen-D4, hydroxybupropion-D6, dextrorphan-D3, 4-hydroxymephenytoin-D3, 1-hydroxymidazolam- 13 C3, N-desethylamodiaquine-D5, and 4-hydroxydiclofenac-D4.

[0017] Specifically, the method comprises the following steps:

[0018] S1. Prepare a series of metabolite stock solutions, a series of probe substrate stock solutions and an internal standard solution;

[0019] S2. Prepare a calibration standard series solution and a quality control sample series solution using a metabolite stock solution series, a probe substrate stock solution series and a potassium phosphate buffer containing microsomes, and establish a calibration curve;

[0020] S3, preparing a double blank sample and a single blank sample, wherein the single blank sample is a potassium phosphate buffer containing microsomes, and the double blank sample is prepared by adding acetonitrile solution to the potassium phosphate buffer containing microsomes;

[0021] S4. Except for the double blank samples, internal standard solution and acetonitrile solution were added to the remaining samples. All samples were vortexed and centrifuged. Then, the supernatant was diluted with pure water and vortexed again for sampling and testing.

[0022] S5. Load the sample to be tested for liquid chromatography tandem mass spectrometry detection. The conditions of liquid chromatography are:

[0023] The chromatographic column model is ACQUITY UPLC HSS T3 2.1*50mm, 1.8μm; the mobile phase A is 2% formic acid-water solution, and the mobile phase B is 2% formic acid acetonitrile solution; the flow rate is 0.7mL / min; the gradient elution program is:

[0024] The mobile phase composition at 0 min was 100% A + 0% B, the mobile phase composition at 1 min was 80% A + 20% B, the mobile phase composition at 2.1 min was 40% A + 60% B, the mobile phase composition at 2.2 min was 10% A + 90% B, the mobile phase composition at 2.3 min was 10% A + 90% B, the mobile phase composition at 2.31 min was 100% A + 0% B, and the mobile phase composition at 2.5 min was 100% A + 0% B;

[0025] The mass spectrum ion information is shown in the following table:

[0026] Analyte name Ion pair (m / z) Retention time (min) Declustering voltage(V) Impact voltage(V) Acetaminophen 152.1 / 110.1 1.08 39 23 Hydroxybupropion 256.4 / 238.0 1.63 50 20 Dextrorphan 258.2 / 157.2 1.59 40 55 4-Hydroxymephenytoin 235.1 / 150.2 1.61 30 36 1-Hydroxymidazolam 342.1 / 203.1 1.84 51 35 N-Desethylamodiaquine 328.2 / 283.2 1.30 60 45 4-Hydroxydiclofenac 312.0 / 231.0 2.25 32 29 <![CDATA[Acetaminophen - D4]]> 156.1 / 114.2 1.07 55 23 <![CDATA[Hydroxybupropion - D6]]> 262.3 / 139.0 1.63 31 30 <![CDATA[Dextromoramide-D3]]> 261.3 / 157.0 1.59 37 52 <![CDATA[4-Hydroxymephenytoin-D3]]> 238.2 / 150.2 1.61 37 24 <![CDATA[1-Hydroxy midazolam- 13 C3]]> 342.1 / 203.1 1.84 53 37 <![CDATA[N-Desethyl Amodiaquine-D5]]> 333.3 / 283.2 1.30 60 25 <![CDATA[4-Hydroxy Diclofenac-D4]]> 316.0 / 234.8 2.25 65 25

[0027] The detection parameters of the mass spectrometer are: select electrospray ion source, multiple reaction ion monitoring, positive ion mode; curtain gas is 40psi; nebulizer gas is 55psi; auxiliary heater is 60psi; ion transmission voltage is 5500V; nebulizer temperature is 250℃; collision gas is 8.

[0028] Specifically, the metabolite stock series solutions are prepared from methanol and the metabolites, including 12.0 mM acetaminophen, 10.0 mM hydroxybupropion, 4.00 mM dextrorphan, 4.00 mM 4-hydroxymephenytoin, 5.00 mM 1-hydroxymidazolam, 10.0 mM N-desethylamodiaquine and 10.0 mM 4-hydroxydiclofenac.

[0029] Specifically, the probe substrate stock series solution is prepared from methanol and the probe substrate, including 20.0 mM phenacetin, 80.0 mM bupropion, 10.0 mM dextromethorphan, 20.0 mM S-mephenytoin, 10.0 mM midazolam, 2.00 mM amodiaquine and 10.0 mM diclofenac.

[0030] Specifically, the internal standard solution is prepared by first preparing an internal standard stock solution with methanol and an internal standard substance and mixing them, and then diluting and mixing them with acetonitrile to prepare the internal standard solution. The internal standard solution is a seven-in-one internal standard working solution, including 100nM acetaminophen-D4, 100nM hydroxybupropion-D6, 20nM dextrorphan-D3, 100nM 4-hydroxymephenytoin-D3, 100nM 1-hydroxymidazolam- 13 C3, 360 nM N-desethylamodiaquine-D5 and 700 nM 4-hydroxydiclofenac-D4.

[0031] Specifically, in the potassium phosphate buffer containing microsomes, the concentration of microsomes is 0.2 mg / mL and the concentration of potassium phosphate is 100 mM.

[0032] Specifically, the calibration standard solution series includes an acetaminophen calibration standard solution with a concentration range of 15.0 to 4000 nM, a hydroxybupropion calibration standard solution with a concentration range of 1.5 to 400 nM, an N-desethylamodiaquine calibration standard solution with a concentration range of 6.0 to 1600 nM, a 4-hydroxydiclofenac calibration standard solution with a concentration range of 30.0 to 8000 nM, a 4-hydroxymephenytoin calibration standard solution with a concentration range of 1.2 to 320 nM, a dextrorphan calibration standard solution with a concentration range of 3.0 to 800 nM, and a 1-hydroxymidazolam calibration standard solution with a concentration range of 3.0 to 800 nM.

[0033] Specifically, the vortexing time is 10 minutes, and the centrifugation condition is 4000 rpm and centrifugation at 4° C. for 20 minutes.

[0034] Specifically, the injection volume was 15 μL.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention improves the analysis speed: The method provided by the present invention can simultaneously determine 7 metabolites. In the detection of seven substrates and their corresponding metabolites and candidate drugs, possible interference within the system is eliminated, and the analysis speed is shortened to 2.5 minutes per injection, which is 3 times faster than the existing technology.

[0037] 2. The detection method provided by the present invention is highly scientific and reliable: When evaluating a large number of candidate drugs, in addition to detecting the target product, it is also necessary to evaluate whether the addition of the candidate drug interferes with the product analysis and whether it affects the test results. The prior art requires separate preparation of standard solutions of metabolites and candidate drugs for investigation during evaluation. The method provided in this application uses the isotope internal standard corresponding to the product, which can quickly track and indicate the degree of interference of the product, and can eliminate interference to a certain extent. Therefore, this method greatly improves the scientificity and reliability of the evaluation of a large number of candidate drugs of unknown structure, and greatly simplifies the process of interference evaluation.

[0038] 3. This method saves detection costs: This method reduces the consumption of organic solvents in the mobile phase, saving more than twice the detection cost. In addition, compared with the laboratory 5-in-1 and two single enzymes incubated separately, the 7-in-1 cocktail method saves 67% of human liver microsomes.

[0039] 4. This method can meet the needs of high-throughput detection: on the basis of significantly shortening the analysis time and ensuring the reliability of the analysis method, the analysis and evaluation of high-throughput samples can be carried out smoothly, and more time can be saved for the subsequent steps of drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a typical acquisition diagram of metabolites corresponding to the seven probe substrates;

[0041] Figure 2 This is the spectrum of 4-hydroxymephenytoin before mass spectrometry parameter optimization;

[0042] Figure 3 This is the spectrum of 4-hydroxymephenytoin after mass spectrometry parameters were optimized;

[0043] Figure 4 This is the spectrum before mobile phase optimization;

[0044] Figure 5 This is the spectrum after mobile phase optimization;

[0045] Figure 6 is a calibration curve graph of acetaminophen in the concentration range of 15-4000 nM;

[0046] Figure 7 is a calibration curve graph of hydroxybupropion in the concentration range of 1.5-400 nM;

[0047] Figure 8 is a calibration curve graph of dextrorphan in the concentration range of 3-800 nM;

[0048] Fig. 9 is a calibration curve graph of 4-hydroxymephenytoin in the concentration range of 1.2-320 nM;

[0049] Fig.10 is a calibration curve graph of 1-hydroxymidazolam in the concentration range of 3-800 nM;

[0050] Fig.11 is a calibration curve graph of N-desethylamodiaquine in the concentration range of 6-1600 nM;

[0051] Fig.12 is a calibration curve graph of 4-hydroxydiclofenac in the concentration range of 30-8000 nM;

[0052] Fig.13 The accuracy and precision of 7 metabolites in 100 mM potassium phosphate buffer containing microsomes. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0054] In the following examples, methanol, acetonitrile and formic acid were of chromatographic grade, and the rest were of analytical grade. The information of the reagents, instruments and consumables involved is as follows:

[0055] Acetaminophen (purity>99.9%, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.), hydroxybupropion (purity>95.0%, purchased from Toronto Research Chemicals), dextrorphan (purity>98.5%, purchased from Shanghai Yuansi Biaowu Technology Co., Ltd.), 4-hydroxymephenytoin (purity>99.4%, purchased from MedChemExpress), 1-hydroxymidazolam (purity>98.0%, purchased from Toronto Research Chemicals), N-desethylamodiaquine (purity>98.2%, purchased from Cayman), 4-hydroxydiclofenac (purity>97.0%, purchased from TorontoResearch Chemicals), phenacetin (purity>99.8%, purchased from Shanghai Yuanye Biological), bupropion (purity 100.0%, purchased from TOKYO CHEMICAL), dextromethorphan (purity>99.8%, purchased from Dalian Bogreen Biotechnology Co., Ltd.), S-mephenytoin (purity>99.8%, purchased from Bailingwei Technology Co., Ltd.), midazolam (purity>98.4%, purchased from Shanghai Yuansi Biao Technology Co., Ltd.), amodiaquine (purity 100.0%, purchased from Sigma-Aldrich), diclofenac (purity>99.3%, purchased from Shanghai Aladdin Biotechnology Co., Ltd.), acetaminophen-D4 (purity>99.6%, purchased from CATO Research Chemicals Inc.), hydroxybupropion-D6 (purity>95.0%, purchased from Toronto Research Chemicals), dextrorphan-D3 (purity>98.6%, purchased from Shanghai Yuansi Biao Technology Co., Ltd.), 4-hydroxymephenytoin-D3 (purity>98.0%, purchased from Toronto Research Chemicals), 1-hydroxymidazolam- 13C3 (purity> 96.0%, purchased from Toronto Research Chemicals), N-deethylamodiaquine-D5 (purity> 99.6%, purchased from CATO Research Chemicals Inc.), 4-hydroxydiclofenac-D4 (purity> 98.7%, purchased from CATO Research Chemicals Inc.). Matrix: 100 mM potassium phosphate buffer containing microsomes (0.2 mg / mL) was prepared by Shanghai WuXi AppTec Laboratory. Liquid phase system: Shimadzu LC-40D X3, purchased from Shimadzu Corporation; injection system: CTC PAL3-RSI, purchased from Zhida Corporation; mass spectrometer: SCIEXTriple Quad 6500+, purchased from SCIEX Corporation.

[0056] Embodiment 1

[0057] In this example, a liquid chromatography-mass spectrometry detection method for simultaneously detecting seven key probe substrate metabolites in a CYP inhibition test using this method is shown, and the specific operation is as follows:

[0058] 1. Preparation of stock solution

[0059] Preparation of metabolite stock solutions: Weigh appropriate amounts of reference standards respectively, place in brown glass bottles, and use methanol to prepare 12.0 mM acetaminophen, 10.0 mM hydroxybupropion, 4.00 mM dextrorphan, 4.00 mM 4-hydroxymephenytoin, 5.00 mM 1-hydroxymidazolam, 10.0 mM N-desethylamodiaquine, and 10.0 mM 4-hydroxydiclofenac metabolite stock solutions respectively.

[0060] Preparation of substrate stock solution: Weigh appropriate amounts of reference standards respectively, place in brown glass bottles, and use methanol to prepare substrate stock solutions of 20.0 mM phenacetin, 80.0 mM bupropion, 10.0 mM dextromethorphan, 20.0 mM S-mephenytoin, 10.0 mM midazolam, 2.00 mM amodiaquine, and 10.0 mM diclofenac respectively.

[0061] Preparation of internal standard solution: Weigh appropriate amounts of reference standard substances, place in brown glass bottles, and prepare with methanol to 6.00 mM acetaminophen-D4, 6.40 mM hydroxybupropion-D6, 10.0 mM dextrorphan-D3, 12.8 mM 4-hydroxymephenytoin-D3, 10.0 mM 1-hydroxymidazolam- 13 C3, internal standard stock solutions of 4.80 mM N-desethylamodiaquine-D5, 4.80 mM 4-hydroxydiclofenac-D4.

[0062] The internal standard stock solution was further mixed and diluted with acetonitrile to form a seven-in-one internal standard working solution with concentrations of 100 nM acetaminophen-D4, 100 nM hydroxybupropion-D6, 20 nM dextrorphan-D3, 100 nM 4-hydroxymephenytoin-D3, 100 nM 1-hydroxymidazolam- 13 C3, 360 nM N-desethylamodiaquine-D5 and 700 nM 4-hydroxydiclofenac-D4.

[0063] The corresponding relationships between the seven metabolic enzymes and the probe substrates, metabolites and isotope internal standards are shown in Table 1:

[0064] Table 1

[0065]

[0066] 2. Preparation of interference evaluation samples, calibration standards and quality control samples

[0067] Interference evaluation samples: appropriate amounts of metabolite stock solution and substrate stock solution were prepared with methanol to prepare metabolite intermediate working solution and substrate intermediate working solution of a certain concentration, and the intermediate working solution was further prepared with methanol-water (1:1, v:v) to prepare interference evaluation working solution, wherein the single standard working solution was prepared for 7 metabolites respectively, and the mixed standard working solution contained 7 metabolites and 7 substrates, with concentrations of 500nM acetaminophen, 100 hydroxybupropion, 250nM dextrorphan, 25nM 4-hydroxymephenytoin, 500nM 1-hydroxymidazolam, 700nM N-desethylamodiaquine, 1000nM 4-hydroxydiclofenac, 50000nM phenacetin, 20000nM bupropion, 5000nM dextrorphan, 50000nM S-mephenytoin, 2000nM midazolam, 2000nM amodiaquine, 10000nM diclofenac.

[0068] Calibration standards and quality control samples: Appropriate amounts of metabolite stock solutions and substrate stock solutions were prepared into metabolite intermediate working solutions of a certain concentration using methanol-water (1:1, v:v). Calibration standards and quality control samples were prepared using metabolite intermediate working solutions and 100 mM potassium phosphate buffer containing microsomes (0.2 mg / mL). Six sets of quality control standards were prepared in parallel.

[0069] 3. Sample Pretreatment

[0070] Interference evaluation sample: Take 300 μL of methanol-water (1:1, v:v) as a blank sample, and take 300 μL of the interference evaluation sample and place them in a 96-well plate with an injection volume of 2 μL, and use a mass spectrometer for detection.

[0071] Calibration standards and quality control samples: Take 200 μL of 100 mM potassium phosphate buffer containing microsomes (0.2 mg / mL) as double blank and single blank samples. Add 400 μL of acetonitrile solution to the double blank sample, and add 400 μL of acetonitrile solution containing internal standard to other samples. Vortex the blank sample and the calibration standards and quality control samples under the precision & accuracy item for 10 minutes, and centrifuge the 96-well plate at 4000 rpm and 4°C for 20 minutes. Take 100 μL of the supernatant and place it in a 96-well plate, add 200 μL of pure water to dilute, and vortex for 10 minutes. The injection volume is 15 μL, and the mass spectrometer is used for detection.

[0072] 4. Testing conditions

[0073] The liquid chromatography-tandem mass spectrometry detection conditions are shown in Table 2 below:

[0074] Table 2

[0075]

[0076]

[0077] In this application, the typical acquisition diagrams of metabolites corresponding to the seven probe substrates are shown in Figure 1 shown.

[0078] Embodiment 2

[0079] In this embodiment, the method provided by the present invention is subjected to condition optimization and performance analysis, and its scientificity and reliability, linearity, accuracy and precision are analyzed respectively. The detection conditions of the performance analysis are the same as those in the first embodiment, and are not described in detail here.

[0080] 1. Condition Optimization

[0081] Since the output of the metabolite 4-hydroxymephenytoin is low and its mass spectrometry response is low, it is particularly important to improve the mass spectrometry response of 4-hydroxymephenytoin. The method provided in the present application reduces the atomization temperature from 500°C to 250°C and the collision gas from 10 to 8 by optimizing the mass spectrometry parameters, thereby increasing the signal-to-noise ratio by 5 times. Figure 2 and Figure 3 As shown, Figure 2 This is the spectrum of 4-hydroxymephenytoin before mass spectrometry parameter optimization. Figure 3 This is the spectrum of 4-hydroxymephenytoin after mass spectrometry parameters were optimized. It can be seen that after optimization, the peak height of 4-hydroxymephenytoin can reach 6781cps;

[0082] In addition, the present application significantly improves the peak shape of acetaminophen and N-desethylamodiaquine by increasing the content of formic acid in the mobile phase from 0.1% to 2%, and solves the problem of the peak front, as follows Figure 4and Figure 5 As shown, Figure 4 This is the spectrum before mobile phase optimization. Figure 5 This is the spectrum after mobile phase optimization. It can be seen that after optimization, the 7 metabolites all show good symmetrical peak shapes.

[0083] 2. Interference evaluation

[0084] First, the peak areas of each product in the metabolite single standard and the substrate and metabolite mixed standard were integrated, and the interference was evaluated by calculating the coefficient of variation between the two (n = 3). The interference of all metabolites in methanol-water (1:1, v:v) was within 15%. The results are shown in Table 3 below, indicating that under this analytical condition, the substrate had no obvious interference on each metabolite and between the 7 metabolites.

[0085] Table 3

[0086]

[0087] 3. Calibration curve

[0088] 1 / x 2 The weighted coefficients were used to perform linear regression on the concentrations of seven metabolites in 100 mM potassium phosphate buffer containing microsomes (0.2 mg / mL). The linear range and calibration curve parameters are shown in Table 4. The calibration curves of the seven metabolites are shown in Figures 6-12. The correlation coefficients of the calibration curves of all analytes in the test were r≥0.99. The test results showed that the linearity met the acceptance criteria and the calibration curves were suitable for quantitative analysis.

[0089] Table 4

[0090] Metabolites Slope (a) Intercept(b) Correlation coefficient (r) Concentration range (nM) Acetaminophen 0.01591 -0.03097 0.99748 15.0～4000 Hydroxybupropion 0.07546 -0.01447 0.99757 1.5～400 N-Desethylamodiaquine 0.01781 -0.01352 0.9982 6.0～1600 4-Hydroxydiclofenac 0.00214 -0.00622 0.99775 30.0～8000 4-Hydroxymephenytoin 0.00943 0.00324 0.99607 1.2～320 Dextrorphan 0.05263 -0.03427 0.99609 3.0～800 1-Hydroxymidazolam 0.08433 -0.00133 0.99696 3.0～800

[0091] 4. Accuracy and precision

[0092] The accuracy and precision were evaluated by one analytical batch and six determinations at each quality control sample concentration level, and the results are shown in Table 4. The coefficient of variation (CV, %) of the precision of each analyte was within ±25.0%, and the relative error (BIAS, %) of the accuracy was within ±25.0%. The test results showed that the accuracy and precision met the acceptance criteria and the analytical method was reliable.

[0093] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test, characterized in that: The probe substrates and metabolites of the CYP subtypes to be evaluated in the CYP inhibition test are determined, and then the concentrations of all metabolites are simultaneously detected using a liquid chromatography tandem mass spectrometry detection method, wherein the CYP subtypes to be evaluated are CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, wherein: The probe substrate of CYP1A2 is phenacetin, and the metabolite is acetaminophen; The probe substrate of CYP2B6 is bupropion, and the metabolite is hydroxybupropion; The probe substrate for CYP2C8 is amodiaquine, and the metabolite is N-desethylamodiaquine; The probe substrate of CYP2C9 is diclofenac, and the metabolite is 4-hydroxydiclofenac; The probe substrate for CYP2C19 is S-mephenytoin, and the metabolite is 4-hydroxymephenytoin; The probe substrate of CYP2D6 is dextrorphan, and the metabolite is dextrorphan; The probe substrate of CYP3A is midazolam and its metabolite is 1-hydroxymidazolam.

2. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 1, characterized in that: The liquid chromatography tandem mass spectrometry detection is an internal standard method, and the internal standard substances are acetaminophen-D4, hydroxybupropion-D6, dextrorphan-D3, 4-hydroxymephenytoin-D3, 1-hydroxymidazolam- 13 C3, N-desethylamodiaquine-D5, and 4-hydroxydiclofenac-D4.

3. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 1, characterized in that: The method comprises the following steps: S1. Prepare a series of metabolite stock solutions, a series of probe substrate stock solutions and an internal standard solution; S2. Prepare a calibration standard series solution and a quality control sample series solution using a metabolite stock solution series, a probe substrate stock solution series and a potassium phosphate buffer containing microsomes, and establish a calibration curve; S3, preparing a double blank sample and a single blank sample, wherein the single blank sample is a potassium phosphate buffer containing microsomes, and the double blank sample is prepared by adding acetonitrile solution to the potassium phosphate buffer containing microsomes; S4. Except for the double blank samples, internal standard solution and acetonitrile solution were added to the remaining samples. All samples were vortexed and centrifuged. Then, the supernatant was diluted with pure water and vortexed again for sampling and testing. S5. Load the sample to be tested for liquid chromatography tandem mass spectrometry detection. The conditions of liquid chromatography are: The chromatographic column model is ACQUITY UPLC HSS T3 2.1*50mm, 1.8μm; the mobile phase A is 2% formic acid-water solution, and the mobile phase B is 2% formic acid acetonitrile solution; the flow rate is 0.7mL / min; the gradient elution program is: The mobile phase composition at 0 min was 100% A + 0% B, the mobile phase composition at 1 min was 80% A + 20% B, the mobile phase composition at 2.1 min was 40% A + 60% B, the mobile phase composition at 2.2 min was 10% A + 90% B, the mobile phase composition at 2.3 min was 10% A + 90% B, the mobile phase composition at 2.31 min was 100% A + 0% B, and the mobile phase composition at 2.5 min was 100% A + 0% B; The mass spectrum ion information is shown in the following table: The detection parameters of the mass spectrometer are: select electrospray ion source, multiple reaction ion monitoring, positive ion mode; curtain gas is 40psi; nebulizer gas is 55psi; auxiliary heater is 60psi; ion transmission voltage is 5500V; nebulizer temperature is 250℃; collision gas is 8.

4. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The metabolite stock series solutions are prepared from methanol and the metabolites, including 12.0 mM acetaminophen, 10.0 mM hydroxybupropion, 4.00 mM dextrorphan, 4.00 mM 4-hydroxymephenytoin, 5.00 mM 1-hydroxymidazolam, 10.0 mM N-desethylamodiaquine and 10.0 mM 4-hydroxydiclofenac.

5. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The probe substrate stock series solution is prepared from methanol and the probe substrate, and includes 20.0 mM phenacetin, 80.0 mM bupropion, 10.0 mM dextromethorphan, 20.0 mM S-mephenytoin, 10.0 mM midazolam, 2.00 mM amodiaquine and 10.0 mM diclofenac.

6. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The internal standard solution is prepared by first preparing an internal standard stock solution with methanol and an internal standard substance, mixing the solution, and then diluting and mixing the solution with acetonitrile. The internal standard solution is a seven-in-one internal standard working solution, including 100nM acetaminophen-D4, 100nM hydroxybupropion-D6, 20nM dextrorphan-D3, 100nM 4-hydroxymephenytoin-D3, 100nM 1-hydroxymidazolam- 13 C3, 360 nM N-desethylamodiaquine-D5 and 700 nM 4-hydroxydiclofenac-D4.

7. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: In the potassium phosphate buffer containing microsomes, the concentration of microsomes is 0.2 mg / mL and the concentration of potassium phosphate is 100 mM.

8. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The calibration standard sample series solutions include a paracetamol calibration standard sample solution with a concentration range of 15.0 to 4000 nM, a hydroxybupropion calibration standard sample solution with a concentration range of 1.5 to 400 nM, an N-desethylamodiaquine calibration standard sample solution with a concentration range of 6.0 to 1600 nM, a 4-hydroxydiclofenac calibration standard sample solution with a concentration range of 30.0 to 8000 nM, a 4-hydroxymephenytoin calibration standard sample solution with a concentration range of 1.2 to 320 nM, a dextrorphan calibration standard sample solution with a concentration range of 3.0 to 800 nM, and a 1-hydroxymidazolam calibration standard sample solution with a concentration range of 3.0 to 800 nM.

9. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The vortexing time was 10 minutes, and the centrifugation condition was 4000 rpm and centrifugation at 4° C. for 20 minutes.

10. The liquid chromatography-mass spectrometry detection method for simultaneously determining seven key probe substrate metabolites in a CYP inhibition test according to claim 3, characterized in that: The injection volume was 15 μL.

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