A method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma

Abstract

The present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, comprising: adding an ammonium acetate solution to a human plasma sample to precipitate proteins in the plasma sample, then adding acetonitrile containing an internal standard solution for extraction, using a high performance liquid chromatography-mass spectrometry method to determine the obtained sample solution, determining the twelve tyrosine kinase inhibitors in the sample solution according to the retention time, and using an internal standard standard curve method for quantitative determination to determine the concentrations of the twelve tyrosine kinase inhibitors in the sample solution. The method provided by the present invention for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma has the advantages of being simple and rapid, having a high analytical throughput, high sensitivity, strong specificity, good precision and accuracy, good stability, high extraction recovery rate, and no obvious matrix effect and dilution effect, and can be used for monitoring the blood drug concentration of tyrosine kinase inhibitors, which are commonly used anti-tumor drugs in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical blood drug concentration monitoring, and relates to a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, and specifically relates to a high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma. Background Art

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality rate in the world. Non-small-cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases, and the number of deaths from lung cancer is about 1.5 million each year. NSCLC is highly malignant and prone to recurrence and metastasis. Most patients are in the advanced stage when diagnosed. Nowadays, personalized molecular targeted therapy based on molecular targets has become a hot topic in NSCLC research. Small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib, afatinib, dacomitinib, icotinib, osimertinib, crizotinib, alectinib, ceritinib, trametinib, and dabrafenib have been used as first-line treatments for NSCLC-related gene mutations. Anlotinib is currently used as a third-line treatment for NSCLC.

[0003] All tyrosine kinase inhibitors are administered orally, which is convenient and improves the quality of life. Another advantage of oral administration is that tyrosine kinase inhibitors are usually taken continuously every day (compared to the intermittent use of most chemotherapy), which generally increases the time that the tumor is exposed to the active drug. However, while it brings convenience to patients, all patients have the same starting dose and show large inter- and intra-individual differences. Most of the first-line small molecule targeted therapeutic drugs for patients with advanced NSCLC are metabolized by the liver drug enzyme CYP450, especially CYP3A4. Pharmacokinetic differences may lead to differences in blood drug concentration exposure in patients, resulting in some patients with too high or too low blood drug concentrations, thus affecting the efficacy. Many studies have pointed out that patients lack compliance with treatment, and compliance is correlated with treatment efficacy. Lack of compliance, as well as increased drug-drug interactions (DDI) or specific pharmacokinetic characteristics through the oral route, may lead to insufficient dosing, reduced efficacy, and even drug resistance. On the contrary, due to DDI, drug overdose caused by weakened metabolism in old age may lead to increased drug toxicity, such as severe rash caused by EGFR inhibitors. Considering the prognosis of the disease, the wide pharmacokinetic variability of drugs, long-term use, and the high cost of drugs, therapeutic drug monitoring (TDM) may be a very useful tool to assist clinicians in making individual dose adjustments.

[0004] So far, researchers have used different methods to measure the concentrations of the above drugs, mainly high-performance liquid chromatography and mass spectrometry. For example, the LC-MS / MS method developed by Essam Ezzeldin et al. was used to detect seven tyrosine kinase inhibitors including gefitinib and osimertinib, using a pretreatment method of ethyl acetate-tert-butyl methyl ether (1:1) liquid-liquid extraction (see: CEEAB, BMIA, ARNH, et al. Simultaneous quantitative determination of seven novel tyrosine kinase inhibitors in plasma by a validated UPLC-MS / MS method and its application to human microsomal metabolic stability study [J]. Journal of Chromatography B, 2020 Jan1; 1136: 1218-1251); GD Marijn Veerman et al. established an HPLC-MS / MS method for detecting the concentrations of afatinib, alectinib, crizotinib, and osimertinib in plasma, using protein precipitation as the pretreatment method (see: Marijn GD, Veerman, Mei H, et al. Quantification of afatinib,alectinib,crizotinib andosimertinib in human plasma by liquid chromatography / triple-quadrupole massspectrometry; focusing on the stability of osimertinib.[J].Journal ofchromatography.B,Analytical technologies in the biomedical and life sciences,2019 Apr 15;1113:37-44.); Rafael Reis et al. established a quantitative method for detecting the concentrations of afatinib, erlotinib, osimertinib and crizotinib in plasma, and the pretreatment method was protein precipitation (see: Reis R, Labat L, Allard M, Boudou-Rouquette P, Chapron J, Bellesoeur A, Thomas-Schoemann A, Arrondeau J, Giraud F, Alexandre J, Vidal M, Goldwasser F, Blanchet B. Liquid chromatography-tandem mass spectrometric assay for therapeutic drug monitoring of the EGFR inhibitors afatinib, erlotinib and osimertinib, the ALK inhibitor crizotinib and the VEGFR inhibitor nintedanib in human plasma from non-small cell lung cancer patients. J Pharm Biomed Anal. 2018 Sep 5; 158: 174-183.). These testing standards and methods are not uniform, and in clinical practice, different patients may take different targeted drugs at the same time, and may require different methods or even different instruments for testing. This not only increases the workload and wastes experimental resources, but also increases the time patients and doctors have to wait for blood drug concentration results. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, combining the analysis of drug characteristic secondary fragment examples, and simultaneously analyzing the concentrations of gefitinib, erlotinib, afatinib, dacomitinib, icotinib, osimertinib, crizotinib, alectinib, ceritinib, trametinib, dabrafenib and anlotinib in human plasma. The method has short detection time, high sensitivity and accurate quantitative analysis.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma in a first aspect, comprising: adding an ammonium acetate solution to a human plasma sample to precipitate proteins in the plasma sample, then adding acetonitrile containing an internal standard solution for extraction, determining the obtained sample solution by a high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method, determining the 12 tyrosine kinase inhibitors in the sample solution according to the retention time, and determining the concentrations of the 12 tyrosine kinase inhibitors in the sample solution by quantitative determination using an internal standard standard curve method.

[0007] Preferably, the 12 tyrosine kinase inhibitors include gefitinib (CAS No. 184475-35-2), erlotinib (CAS No. 183321-74-6), afatinib (CAS No. 850140-72-6), dacomitinib (CAS No. 1110813-31-4), icotinib (CAS No. 610798-31-7), osimertinib (CAS No. 14213 73-65-0), crizotinib (CAS No. 877399-52-5), alectinib (CAS No. 1256580-46-7), ceritinib (CAS No. 1032900-25-6), trametinib (CAS No. 871700-17-3), dabrafenib (CAS No. 1195765-45-7), anlotinib (CAS No. 1058156-90-3).

[0008] Preferably, after adding the ammonium acetate solution to the human plasma sample, the sample is vortexed for 15-45 seconds, preferably for 30 seconds.

[0009] Preferably, the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 1-10 mol / L. More preferably, the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 5 mol / L.

[0010] Preferably, the volume ratio of the ammonium acetate solution to the human plasma sample is 0.9-1.1:1, preferably 1:1.

[0011] Preferably, the internal standard solution is a methanol solution of voriconazole. The CAS number of the voriconazole is 137234-62-9. The voriconazole has no interference with 12 tyrosine kinase inhibitors.

[0012] More preferably, the concentration of the internal standard solution is 1 mg / mL.

[0013] Preferably, the concentration of the internal standard in the acetonitrile containing the internal standard solution is 10-15 ng / mL, preferably 12 ng / mL.

[0014] Preferably, the volume ratio of the acetonitrile to the human plasma sample is 1.9-2.1:1, preferably 2:1.

[0015] Preferably, the extraction is performed by vortexing first and then centrifuging to obtain the supernatant.

[0016] More preferably, the vortex oscillation time is 2-4 min, preferably 3 min.

[0017] More preferably, the centrifugation time is 9-11 minutes, the centrifugation temperature is 3-5°C, and the centrifugation speed is 10000-15000 g / min.

[0018] Further preferably, the centrifugation time is 10 minutes, the centrifugation temperature is 4° C., and the centrifugation speed is 12000 g / min.

[0019] More preferably, the amount of the supernatant is 90-110 μL, preferably 100 μL.

[0020] Preferably, the sample solution is measured by high performance liquid chromatography-mass spectrometry, comprising the following steps:

[0021] 1) Prepare standard solution: add ammonium acetate solution to blank plasma sample to precipitate protein in plasma sample, add drug solution and acetonitrile containing internal standard solution to extract, and obtain standard solution;

[0022] 2) Sample detection: The sample solution and the standard solution in step 1) were detected by high performance liquid chromatography-mass spectrometry, and the liquid chromatogram of the sample solution was compared with the liquid chromatogram of the standard solution. The common characteristic peaks were identified according to the relative retention time for qualitative analysis, and then the concentrations of the 12 tyrosine kinase inhibitors in the sample solution were determined by the internal standard curve method based on the chromatographic peak areas of the common characteristic peaks.

[0023] More preferably, in step 1), the drug solution is a stock solution of 12 tyrosine kinase inhibitor standards, and the solvent used is methanol and dimethyl sulfoxide in a volume ratio of 3-5:1.

[0024] Further preferably, the solvent is methanol and dimethyl sulfoxide in a volume ratio of 4:1.

[0025] More preferably, in step 1), the concentrations of the 12 tyrosine kinase inhibitors in the drug solution are all 1 mg / mL.

[0026] More preferably, in step 1), the internal standard solution is a methanol solution of voriconazole.

[0027] More preferably, in step 1), the concentration of the internal standard solution is 1 mg / mL.

[0028] More preferably, in step 1), the blank plasma sample is vortexed for 15-45 seconds, preferably 30 seconds after adding the ammonium acetate solution.

[0029] More preferably, in step 1), the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 1-10 mol / L.

[0030] Further preferably, the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 5 mol / L.

[0031] More preferably, in step 1), the volume ratio of the ammonium acetate solution to the blank plasma sample is 0.9-1.1:1, preferably 1:1.

[0032] More preferably, in step 1), the volume ratio of the acetonitrile to the blank plasma sample is 1.9-2.1:1, preferably 2:1.

[0033] More preferably, in step 1), the extraction is performed by vortexing first and then centrifuging to obtain the supernatant.

[0034] Further preferably, the vortex oscillation time is 2-4 min, preferably 3 min.

[0035] Further preferably, the centrifugation time is 9-11 minutes, the centrifugation temperature is 3-5°C, and the centrifugation speed is 10000-15000 g / min.

[0036] Most preferably, the centrifugation time is 10 minutes, the centrifugation temperature is 4° C., and the centrifugation speed is 12000 g / min.

[0037] More preferably, the amount of the supernatant is 90-110 μL, preferably 100 μL.

[0038] More preferably, in step 1), the concentration ranges of the 12 tyrosine kinase inhibitors in the standard solution are: gefitinib 1.25-625 ng / mL; erlotinib 0.5-250 ng / mL; afatinib 1-500 ng / mL; dacomitinib 5-2500 ng / mL; icotinib 0.5-250 ng / mL; osimertinib 1-500 ng / mL; crizotinib 10-2500 ng / mL; alectinib 1-500 ng / mL; ceritinib 2-1000 ng / mL; trametinib 1-500 ng / mL; dabrafenib 0.5-250 ng / mL; anlotinib 0.4-200 ng / mL.

[0039] More preferably, in step 1), the concentration of the internal standard in the standard solution is 10-15 ng / mL, preferably 12 ng / mL.

[0040] Preferably, in the high performance liquid chromatography-mass spectrometry method, the determination conditions of the high performance liquid chromatography (HPLC) are:

[0041] Chromatographic column: C18 column; column temperature: 35-45°C; temperature in the injector is room temperature; injection volume: 15-25 μL; flow rate: 0.5-2.0 mL / min; 2-4:6-8 split after the column, 2-4 / 10 flows into mass spectrometry analysis; mobile phase A is water, containing formic acid with a volume fraction of 0.05-0.15% and ammonium acetate with a volume fraction of 4-6 mmol / L; mobile phase B is acetonitrile; analysis time is 6 min; gradient elution.

[0042] More preferably, the determination conditions of the high performance liquid chromatography (HPLC) are:

[0043] Chromatographic column: Waters XBridge C18 column (inner diameter 4.6×column length 100mm, particle size 3.5μm); column temperature: 40℃; temperature in the injector was 20-30℃; injection volume: 20μL; flow rate: 1mL / min; 3:7 split after the column, 3 / 10 flowed into mass spectrometry analysis; mobile phase A was water containing 0.1% formic acid and 5mmol / L ammonium acetate by volume; mobile phase B was acetonitrile; analysis time was 6min; gradient elution.

[0044] More preferably, the specific procedure of the gradient elution is:

[0045] 0-0.5min, volume ratio of phase A:phase B is 80:20-20:80;

[0046] 0.5-3.5min, volume ratio of phase A:phase B is 20:80-20:80;

[0047] 3.5-3.51min, volume ratio of phase A:phase B is 20:80-80:20;

[0048] 3.51-4.0min, volume ratio of phase A:phase B is 80:20-80:20;

[0049] 4.0-4.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0050] 4.01-4.5min, volume ratio of phase A:phase B is 10:90-10:90;

[0051] 4.5-4.51min, volume ratio of phase A:phase B is 10:90-80:20;

[0052] 4.51-6min, the volume ratio of phase A:phase B is 80:20-80:20.

[0053] Under the above gradient elution procedure, all 12 tyrosine kinase inhibitors can be eluted, the resulting chromatographic peaks are narrower and the monitoring time is shorter.

[0054] Preferably, in the high performance liquid chromatography-mass spectrometry method, the measurement conditions of the mass spectrometry (MS / MS) are:

[0055] Ionization mode: electrospray ion source (H-ESI), positive ion detection mode; ion source temperature: 500℃; curtain gas: 25psi; collision gas: 10psi; nebulizer gas: 50psi; auxiliary gas: 45psi; spray voltage: 5500V; monitoring mode: multiple reaction monitoring (MRM) mode for primary / secondary mass spectrometry analysis. The precursor mass channel (Precursor), product mass channel (Product), declustering potential (DP), collision energy (CE), entrance potential (EP), and exit potential (CXP) of 12 tyrosine kinase inhibitors and internal standards are shown in Table 1.

[0056] The above-mentioned multiple reaction monitoring (MRM) mode selectively screens the parent ions through the quadrupole in the mass spectrometer. The screened ions enter the collision cell for collision fragmentation, and the generated fragment ions are scanned at high resolution. Then, the characteristic secondary fragment ions are selected for targeted quantification, which can effectively enhance selectivity, accuracy and sensitivity. It provides reliable experimental data for individualized treatment of tyrosine kinase inhibitors in clinical practice, feedbacks abnormal results, assists in adjusting clinical medication, and avoids the occurrence of adverse reactions while ensuring the efficacy of the drug.

[0057] More preferably, the precursor mass channels (precursor, m / z) of the 12 tyrosine kinase inhibitors and the internal standard are: gefitinib 447.4; erlotinib 394.1; afatinib 486.1; dacomitinib 470.4; osimertinib 500.5; icotinib 392.3; crizotinib 450.3; ceritinib 558.4; alectinib 483.2; trametinib 616.2; dabrafenib 520.2; anlotinib 408.3; voriconazole 350.2, see Figure 1 .

[0058] More preferably, the product ion mass channels (Product, m / z) of the 12 tyrosine kinase inhibitors and the internal standard are: gefitinib 127.8; erlotinib 336.2; afatinib 371.1; dacomitinib 319.1; osimertinib 72.2; icotinib 304.3; crizotinib 259.3; ceritinib 433.2; alectinib 396.3; trametinib 491.1; dabrafenib 307.3; anlotinib 339.3; voriconazole 281.2, see Figure 1 .

[0059] More preferably, the declustering potentials (DP, V) of the 12 tyrosine kinase inhibitors and the internal standard are: gefitinib 100; erlotinib 130; afatinib 112; dacomitinib 100; osimertinib 108; icotinib 75; crizotinib 111; ceritinib 107; alectinib 60; trametinib 142; dabrafenib 145; anlotinib 90; voriconazole 90.

[0060] More preferably, the collision energy (CE, V) of the 12 tyrosine kinase inhibitors and the internal standard are: gefitinib 42; erlotinib 35; afatinib 40; dacomitinib 48; osimertinib 42; icotinib 40; crizotinib 35; ceritinib 67; alectinib 35; trametinib 58; dabrafenib 45; anlotinib 33; voriconazole 27.

[0061] More preferably, the entrance potentials (EP, V) of the 12 tyrosine kinase inhibitors and the internal standard are all 10V.

[0062] More preferably, the exit voltages (Collision Exit Potential, CXP, V) of the 12 tyrosine kinase inhibitors and the internal standard are all 15V.

[0063] Table 1

[0064]

[0065] Preferably, in step 2), the internal standard curve method comprises the following steps:

[0066] A1) performing high performance liquid chromatography-mass spectrometry analysis on standard solutions containing a series of 12 tyrosine kinase inhibitors at different concentrations, respectively, extracting characteristic secondary fragment ions of the 12 tyrosine kinase inhibitor components and the internal standard, obtaining a linear relationship between the ratio of the chromatographic peak area of ​​the 12 tyrosine kinase inhibitors at different concentrations / the chromatographic peak area of ​​the internal standard and the ratio of the different concentrations of the corresponding components / the concentration of the internal standard, plotting a corresponding standard curve, performing regression calculation using a weighted least squares method, and obtaining a regression equation for the standard curve of the 12 tyrosine kinase inhibitor components;

[0067] A2) analyzing the sample solution by high performance liquid chromatography-mass spectrometry, substituting the obtained chromatographic peak area ratios of various tyrosine kinase inhibitor components to the internal standard into the regression equation of the standard working curve of the corresponding component in step A1), and calculating the concentration of the corresponding tyrosine kinase inhibitor component in the sample solution based on the known concentration of the internal standard.

[0068] More preferably, in step A1) or A2), in the standard working curve, the chromatographic peak area ratio of the 12 tyrosine kinase inhibitor components / internal standard is used as the ordinate (Y-axis), and the concentration ratio of the corresponding components to the internal standard is used as the abscissa (X-axis).

[0069] The second aspect of the present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, and its use in monitoring blood drug concentrations in clinical samples.

[0070] Preferably, the blood drug is an anti-tumor drug tyrosine kinase inhibitor.

[0071] As described above, the method provided by the present invention for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma has the following beneficial effects:

[0072] (1) The present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, which is applicable to and meets the detection requirements of twelve different drugs, can significantly reduce subsequent development costs, and is conducive to the unification of detection standards, thereby providing the possibility for data collected at different detection points or laboratories to be mutually referenced or used.

[0073] (2) The present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma. Before analysis by high performance liquid chromatography-mass spectrometry, the sample to be tested is pretreated by salting-out assisted homogeneous liquid-liquid extraction, thereby achieving a higher recovery rate and avoiding matrix effects.

[0074] (3) The method provided by the present invention for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma has the advantages of high sensitivity, strong specificity, good precision and accuracy, and good stability after methodological verification; and the extraction recovery rate of each drug is within an acceptable range, and no obvious matrix effect or dilution effect is observed. At the same time, the present invention also verifies the hemolytic effect. The present invention has the advantages of being simple, rapid, and having a high throughput.

[0075] (4) The present invention provides a method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma. The results of the determination of drug concentrations in clinical plasma samples further prove that the method of the present invention is suitable for the determination of drug concentrations in clinical plasma and can be used for monitoring the blood concentrations of tyrosine kinase inhibitors, which are commonly used anti-tumor drugs in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The secondary ion patterns A, B, C, D, E, F, G, H, I, J, K, L, and M of the twelve tyrosine kinase inhibitors and the internal standard in human plasma of the present invention are shown in Figure 2, wherein A is gefitinib; B is erlotinib; C is afatinib; D is dacomitinib; E is osimertinib; F is icotinib; G is crizotinib; H is ceritinib; I is alectinib; J is trametinib; K is dabrafenib; L is anlotinib; and M is voriconazole. DETAILED DESCRIPTION

[0077] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0078] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0079] The reagents and instruments used in the following examples without indicating the manufacturer are all conventional products available on the market.

[0080] Example 1

[0081] 1. Sample pretreatment

[0082] Methanol was used as a solvent to prepare an internal standard solution, which was a 1 mg / mL methanol solution of voriconazole. The internal standard solution was stored in a -40°C refrigerator.

[0083] Take 100 μL of human plasma sample and place it in a 1.5 mL EP tube. Add 100 μL of 5 mol / L ammonium acetate aqueous solution and vortex for 30 seconds to precipitate the protein in the plasma sample. Then add 200 μL of acetonitrile containing internal standard solution and vortex for 3 minutes. Centrifuge at 12000 g / min for 10 minutes at 4°C. Take 100 μL of supernatant and place it in the injection tube to obtain the sample solution for standby use. The concentration of the internal standard in the acetonitrile containing the internal standard solution is 12 ng / mL.

[0084] 2. Standard solution

[0085] The drug solution was prepared by using methanol and dimethyl sulfoxide in a volume ratio of 4:1 as solvent. The concentration of the 12 tyrosine kinase inhibitors in the drug solution was 1 mg / mL. The drug solution was stored in a -40°C refrigerator.

[0086] Take a series of 100 μL blank plasma samples and place them in 1.5 mL EP tubes. Add 100 μL of 5 mol / L ammonium acetate aqueous solution and vortex for 30 seconds to precipitate the protein in the plasma sample. Then add a series of drug solutions containing 12 tyrosine kinase inhibitors with different concentrations and acetonitrile containing internal standard solution and vortex for 3 minutes. Centrifuge at 12000 g / min for 10 minutes at 4°C. Take 100 μL of supernatant and place it in the injection tube to obtain a series of standard solutions containing 12 tyrosine kinase inhibitors with different concentrations for standby use. The concentration of the internal standard in the standard solution is 12 ng / mL. The series of concentrations of the 12 tyrosine kinases contained in the standard solution are shown in Table 2.

[0087] Table 2

[0088]

[0089]

[0090] 3. Detection

[0091] A series of standard solutions containing 12 tyrosine kinase inhibitors at different concentrations were tested by HPLC-MS / MS. The liquid chromatograms of the sample solutions were compared with those of the standard solutions. The common characteristic peaks were identified and qualitatively determined based on the relative retention times to determine the 12 tyrosine kinase inhibitors in the sample solutions.

[0092] The characteristic secondary fragment ions of the 12 tyrosine kinase inhibitor components and the internal standard were then extracted to obtain the linear relationship between the ratio of the chromatographic peak area of ​​the 12 tyrosine kinase inhibitors at different concentrations / the chromatographic peak area of ​​the internal standard and the ratio of the different concentrations of the corresponding components / the concentration of the internal standard. The corresponding standard curves were drawn, and the weighted least squares method was used for regression calculation to obtain the regression equations of the standard curves of the 12 tyrosine kinase inhibitor components.

[0093] The sample solution was analyzed by high performance liquid chromatography-mass spectrometry, and the chromatographic peak area ratios of various tyrosine kinase inhibitor components and the internal standard were substituted into the regression equation of the standard working curve of the corresponding component, and the content of the corresponding tyrosine kinase inhibitor component in the sample solution was calculated based on the known concentration of the internal standard.

[0094] Among them, the determination conditions of high performance liquid chromatography (HPLC) are as follows: chromatographic column: Waters XBridge C18 column (inner diameter 4.6×column length 100mm, particle size 3.5μm); column temperature: 40°C; temperature in the injector is 20-30°C; injection volume: 20μL; flow rate: 1mL / min; 3:7 split after the column, 3 / 10 flows into mass spectrometry analysis; mobile phase A is water, containing 0.1% by volume formic acid and 5mmol / L ammonium acetate; mobile phase B is acetonitrile; analysis time is 6min; gradient elution.

[0095] The specific procedure of gradient elution is:

[0096] 0-0.5min, volume ratio of phase A:phase B is 80:20-20:80;

[0097] 0.5-3.5min, volume ratio of phase A:phase B is 20:80-20:80;

[0098] 3.5-3.51min, volume ratio of phase A:phase B is 20:80-80:20;

[0099] 3.51-4.0min, volume ratio of phase A:phase B is 80:20-80:20;

[0100] 4.0-4.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0101] 4.01-4.5min, volume ratio of phase A:phase B is 10:90-10:90;

[0102] 4.5-4.51min, volume ratio of phase A:phase B is 10:90-80:20;

[0103] 4.51-6min, the volume ratio of phase A:phase B is 80:20-80:20.

[0104] The determination conditions of mass spectrometry (MS / MS) were as follows: ionization mode: electrospray ion source (H-ESI), positive ion detection mode; ion source temperature was 500°C; curtain gas was 25psi; collision gas was 10psi; nebulizer gas was 50psi; auxiliary gas was 45psi; spray voltage was 5500V; monitoring mode was primary / secondary mass spectrometry analysis in multiple reaction monitoring (MRM) mode; precursor ion mass channel (Precursor), product ion mass channel (Product), declustering potential (DP), collision energy (CE), entrance potential (EP), and exit potential (CXP) of 12 tyrosine kinase inhibitors and internal standards are shown in Table 1.

[0105] Example 2

[0106] A series of blank plasma samples were placed in 1.5 mL EP tubes and processed using step 2 of Example 1 to prepare quality control solutions of 12 tyrosine kinase inhibitors with different quality control concentrations. The concentrations of the 12 tyrosine kinases in the quality control solutions are shown in Table 3.

[0107] Table 3

[0108]

[0109] Example 3

[0110] The method of the present invention was validated by methodology, mainly including linearity, precision and accuracy, matrix effect and recovery rate, dilution effect, hemolytic effect, and stability.

[0111] 1. Linear

[0112] According to Table 2 in step 2 of Example 1, a series of standard solutions containing 12 tyrosine kinase inhibitors of different concentrations were prepared, and the detection was performed according to step 3 of Example 1. The chromatographic peak area ratio of the 12 tyrosine kinase inhibitor components / internal standard was used as the ordinate (Y axis), and the concentration ratio of the corresponding component to the internal standard was used as the abscissa (X axis). The regression equation of the standard curve of the 12 tyrosine kinase inhibitor components was obtained, and the results are shown in Table 4. As can be seen from Table 4, the 12 tyrosine kinase inhibitor components have a good linear relationship, and the correlation coefficient (r 2 ) are all greater than 0.99. The results of the quantitative lower limit are also shown in Table 4.

[0113] Table 4

[0114]

[0115] 2. Precision and Accuracy

[0116] The quality control solution prepared in Example 2 was taken, and 6 portions of each tyrosine kinase inhibitor component and each concentration were prepared, and one batch of samples was prepared every day for 3 consecutive days, and a total of 3 batches were prepared. The test was performed according to step 3 of Example 1, and the intra-batch and inter-batch precision (the absolute value was less than 15% for passing) and accuracy (85-115% for passing) were calculated. The specific results are shown in Table 5.

[0117] As shown in Table 5, the accuracy of each test concentration of each tyrosine kinase inhibitor component in the quality control solution sample is within the range of 85-115%, and the absolute values ​​of intra-batch and inter-batch precision of each test concentration of each tyrosine kinase inhibitor component in the quality control solution sample are less than 15%. This proves that the method of the present invention has good precision and accuracy for the detection of 12 tyrosine kinase inhibitor components in plasma.

[0118] Table 5

[0119]

[0120]

[0121] 3. Matrix Effect and Recovery

[0122] Six human plasma samples from different sources were taken and processed to obtain blank plasma matrices. Six quality control solutions of the 12 tyrosine kinase inhibitor components with known low, medium and high concentrations were prepared with the blank matrix according to the above Example 2. LC-MS / MS detection was performed according to step 3 of Example 1, and the matrix effects and recoveries of the 12 tyrosine kinase inhibitor components were calculated. The results are shown in Table 6. As can be seen from Table 6, the matrix effect and recovery of the method of the present invention are good, and the influence of the plasma matrix on the above twelve drugs and internal standards is basically the same.

[0123] Table 6 Matrix effect and extraction recovery test in human plasma (n=6)

[0124]

[0125]

[0126] 4. Dilution effect

[0127] Sample solutions were prepared according to the concentration of 7 of the 12 tyrosine kinase inhibitor components listed in Table 2, and 6 portions of each sample solution were prepared. The blank plasma was diluted ten-fold or twenty-fold, and the solution was treated according to step 2 of the above-mentioned Example 1, and the solution was tested according to step 3 of Example 1 to calculate the concentration of the 12 tyrosine kinase inhibitor components in the diluted solution. The concentration results were multiplied by 10 or 20 to calculate the concentration of the 12 tyrosine kinase inhibitor components in the undiluted sample solution.

[0128] The calculated results were compared with the concentration 7 in Table 2. The results showed that after 10-fold or 20-fold dilution, the precision of each drug was within 15%, and the accuracy was between 85-115%, indicating that under this dilution condition, there was no obvious dilution effect and the precision and accuracy of the sample were not affected.

[0129] 5. Hemolytic effect

[0130] Blank plasma was repeatedly frozen and thawed twice at -80°C, vortexed vigorously for 3 minutes, and centrifuged at 12000g for 10 minutes. 0.5, 1, 2, 3, 5, 10, 20, and 40 μL of the upper hemolyzed plasma were added to the blank plasma to prepare 0%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, and 4% hemolyzed plasma samples. The 4% hemolyzed plasma sample was used to prepare the quality control samples of concentration 1 and concentration 4 in Table 3 according to the above Example 2, and then tested according to step 3 of Example 1. The precision of the 12 tyrosine kinase inhibitor components was within 15%, and the accuracy was between 85-115%, indicating that under the hemolytic conditions, no obvious hemolytic effect occurred, and the precision and accuracy of the samples were not affected.

[0131] 6. Stability

[0132] Plasma samples under different conditions were investigated, including 6 hours at room temperature, 30-day stability, freeze-thaw three times, and 24 hours of sample injection after treatment. The plasma samples were pre-treated according to step 1 of Example 1 and tested according to step 3 of Example 1. The results are shown in Table 7. As can be seen from Table 7, the RE values ​​of the 12 tyrosine kinase inhibitor components in the plasma samples under the above-mentioned investigation conditions were all less than 15%. Under the above-mentioned investigation conditions, the method of the present invention has strong stability in detecting the above 12 tyrosine kinase inhibitor components in plasma.

[0133] Table 7

[0134]

[0135]

[0136] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma, comprising: An ammonium acetate solution is added to a human plasma sample to precipitate proteins in the plasma sample, and then acetonitrile containing an internal standard solution is added for extraction. The obtained sample solution is measured by high performance liquid chromatography-mass spectrometry, and the 12 tyrosine kinase inhibitors in the sample solution are determined according to the retention time. The internal standard standard curve method is used for quantitative determination to determine the concentrations of the 12 tyrosine kinase inhibitors in the sample solution; The 12 tyrosine kinase inhibitors consist of gefitinib, erlotinib, afatinib, dacomitinib, icotinib, osimertinib, crizotinib, alectinib, ceritinib, trametinib, dabrafenib and anlotinib; The internal standard solution is a methanol solution of voriconazole; The sample solution is measured by high performance liquid chromatography-mass spectrometry, comprising the following steps: 1) Prepare standard solution: add ammonium acetate solution to blank plasma sample to precipitate protein in plasma sample, add drug solution and acetonitrile containing internal standard solution to extract, and obtain standard solution; 2) Sample detection: The sample solution and the standard solution in step 1) are detected by high performance liquid chromatography-mass spectrometry, respectively, and the liquid chromatogram of the sample solution is obtained and compared with the liquid chromatogram of the standard solution, and the common characteristic peaks are identified according to the relative retention time for qualitative analysis, and then the concentrations of the 12 tyrosine kinase inhibitors in the sample solution are determined by the internal standard curve method based on the chromatographic peak areas of the common characteristic peaks; In step 1), the drug solution is a stock solution of 12 tyrosine kinase inhibitor standards, and the solvent used is methanol and dimethyl sulfoxide in a volume ratio of 3-5:1; In step 2), in the HPLC-MS method, the HPLC determination conditions are as follows: chromatographic column: C18 column; column temperature: 35-45°C; temperature in the injector is room temperature; injection volume: 15-25 μL; flow rate: 0.5-2.0 mL / min; 2-4:6-8 split after the column, 2-4 / 10 flows into mass spectrometry analysis; mobile phase A is water, containing formic acid with a volume fraction of 0.05-0.15% and ammonium acetate with a volume fraction of 4-6 mmol / L; mobile phase B is acetonitrile; analysis time is 6 min; gradient elution; The specific procedure of the gradient elution is: 0-0.5min, the volume ratio of phase A:phase B is 80:20-20:80; 0.5-3.5min, the volume ratio of phase A:phase B is 20:80-20:80; 3.5-3.51min, the volume ratio of phase A:phase B is 20:80-80:20; 3.51-4.0min, the volume ratio of phase A: phase B is 80:20-80:20; 4.0-4.01min, the volume ratio of phase A: phase B is 80:20-10:90; 4.01-4.5min, the volume ratio of phase A: phase B is 10:90-10:90; 4.5-4.51min, the volume ratio of phase A: phase B is 10:90-80:20; 4.51-6min, the volume ratio of phase A: phase B is 80:20-80:20; In step 2), in the high performance liquid chromatography-mass spectrometry method, the mass spectrometry measurement conditions are as follows: ionization mode: electrospray ionization source H-ESI, positive ion detection mode; ion source temperature is 500°C; curtain gas is 25psi; collision gas is 10psi; nebulizer gas is 50psi; auxiliary gas is 45psi; spray voltage is 5500V; monitoring mode is multiple reaction monitoring MRM mode for primary / secondary mass spectrometry analysis; 2. The method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma according to claim 1, characterized in that: The ammonium acetate solution precipitates proteins in the plasma sample, including any one or more of the following conditions: A1) After adding the ammonium acetate solution to the human plasma sample, vortex oscillation was performed for 15-45 seconds; A2) the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 1-10 mol / L; A3) The volume ratio of the ammonium acetate solution to the human plasma sample is 0.9-1.1:

1.

3. The method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma according to claim 1, characterized in that: The adding of acetonitrile containing an internal standard solution for extraction comprises any one or more of the following conditions: B1) the concentration of the internal standard in the acetonitrile containing the internal standard solution is 10-15 ng / mL; B2) the volume ratio of the acetonitrile to the human plasma sample added is 1.9-2.1:1; B3) The extraction is performed by vortex oscillation first and then centrifugation to take the supernatant; the vortex oscillation time is 2-4 minutes; the centrifugation time is 9-11 minutes, the centrifugation temperature is 3-5°C, and the centrifugation speed is 10000-15000g / min.

4. The method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma according to claim 1, characterized in that: In step 1), any one or more of the following conditions are included: C1) the internal standard solution is a methanol solution of voriconazole; C2) the blank plasma sample was vortexed for 15-45 seconds after adding the ammonium acetate solution; C3) the ammonium acetate solution is an aqueous solution of ammonium acetate with a concentration of 1-10 mol / L; C4) the volume ratio of the ammonium acetate solution to the blank plasma sample is 0.9-1.1:1; C5) the volume ratio of the acetonitrile to the blank plasma sample is 1.9-2.1:1; C6) The extraction is performed by vortex oscillation first and then centrifugation to take the supernatant; the vortex oscillation time is 2-4 minutes; the centrifugation time is 9-11 minutes, the centrifugation temperature is 3-5°C, and the centrifugation speed is 10000-15000g / min.

5. Use of the method for simultaneously determining the concentrations of twelve tyrosine kinase inhibitors in human plasma according to any one of claims 1 to 4 in monitoring blood drug concentrations in clinical samples.

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