A method and kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS

Abstract

The present invention discloses a method and a kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS. First, working solutions of olverembatinib standard curves with different concentrations and working solutions of olverembatinib quality controls with different concentrations are prepared. Then, an internal standard working solution is configured. Finally, the internal standard working solution is added to the sample to be tested, vortexed and mixed, and then pretreated to obtain a supernatant. The supernatant is taken and injected into an LC-MS / MS instrument for detection and analysis. The present invention not only has simple pretreatment, low cost, short analysis time, and good specificity, but also has high sensitivity of the detection method, and the lower limit of quantification can reach the picogram level, which is rare in other LC-MS / MS detection methods. This method can be used for the pharmacokinetic-pharmacodynamic study of olverembatinib and the determination of the concentration of olverembatinib in the plasma and cerebrospinal fluid of conventional clinical patients.

Description

Technical Field

[0001] The present invention relates to the field of drug concentration detection and analysis, and particularly to a method and kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS. Background Art

[0002] Olverembatinib (trade name: Nairike, English name: Olverembatinib) is a first-class new drug developed in China. It was launched in November 2021. It is an oral third-generation tyrosine kinase BCR-ABL inhibitor and is the first approved third-generation BCR-ABL targeted drug for the treatment of drug-resistant chronic myeloid leukemia in China. At present, drug resistance during the treatment with first-generation (imatinib) and second-generation (nilotinib, dasatinib, etc.) tyrosine kinase drugs is a common problem, and the T315I mutation is the main cause of drug resistance. Olverembatinib has outstanding effects on various BCR-ABL mutants including the T315I mutation.

[0003] Since olverembatinib has been on the market for a short time, its pharmacokinetics-pharmacodynamics in the human body, especially in patients, is still unclear, which limits its clinical application. Detecting the drug concentration in blood or other biological samples is the basis for pharmacokinetics, pharmacodynamics research and clinical precision treatment. At present, there are no relevant literature and patent reports on the detection of olverembatinib concentration at home and abroad, which hinders the research on olverembatinib-related drug treatment. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method and kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS. This method has the advantages of being fast, simple, sensitive, high recovery rate, and small matrix effect; it does not require expensive reagents and is easy to operate, and can quickly, sensitively and robustly determine the concentration of olverembatinib in plasma and cerebrospinal fluid; it provides a basis for the pharmacokinetics, pharmacodynamics and precision treatment research of olverembatinib.

[0005] To achieve the above purpose, the present invention designs a method for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS, including the following steps:

[0006] 1) Prepare olverembatinib standard curve working solutions with different concentrations and olverembatinib quality control working solutions with different concentrations;

[0007] 2) Prepare an internal standard working solution;

[0008] 3) Add the internal standard working solution to the sample to be tested, vortex mix, then perform pretreatment to obtain a supernatant, and inject the supernatant into an LC-MS / MS instrument for detection and analysis.

[0009] Further, in step 2), the internal standard is any one of clozapine, deuterated imatinib, and deuterated olverembatinib.

[0010] Still further, the sample to be measured is a plasma sample to be measured or a cerebrospinal fluid sample to be measured.

[0011] Still further, the pretreatment steps for the plasma sample to be measured are as follows:

[0012] Add the internal standard working solution to the plasma sample to be measured, vortex mix, and then add methyl tert-butyl ether (MTBE) and vortex; then centrifuge at high speed at low temperature, take the upper organic solvent and dry it with nitrogen, then re-dissolve it with the reconstitution solution, vortex mix, and centrifuge at high speed at low temperature to obtain the supernatant; wherein, the volume of methyl tert-butyl ether (MTBE) is 5-20 times that of the plasma sample to be measured;

[0013] Alternatively, the pretreatment steps for the cerebrospinal fluid sample to be measured are as follows:

[0014] Add the internal standard working solution to the cerebrospinal fluid sample to be measured, vortex mix, and then centrifuge at high speed at low temperature, take the upper organic solvent and dry it with nitrogen, then re-dissolve it with the reconstitution solution, vortex mix, and centrifuge at high speed at low temperature to obtain the supernatant. The volume of the internal standard working solution is 2-10 times that of the cerebrospinal fluid sample to be measured.

[0015] Still further, the internal standard is deuterated imatinib d8; and when pretreating the plasma sample to be measured, the internal standard working solution is diluted with methanol and the concentration is 20 ng / mL;

[0016] Alternatively, the internal standard is deuterated imatinib d8; when pretreating the cerebrospinal fluid sample to be measured, the internal standard working solution is diluted with methanol and the concentration is 70 pg / mL.

[0017] Still further, the reconstitution solution is composed of methanol or acetonitrile and water mixed at a volume ratio of 50:50 to 90:10.

[0018] Preferably, the reconstitution solution is composed of methanol or acetonitrile and water mixed at a volume ratio of 75:25.

[0019] Still further, the chromatographic conditions of the LC-MS / MS instrument are as follows:

[0020] Using a universal C18 silica gel column as the chromatographic column,

[0021] Mobile phase A is an aqueous solution containing ammonium formate or an aqueous solution containing ammonium acetate or 0.5% ammonia water;

[0022] Mobile phase B is methanol, acetonitrile, a mixed solvent of methanol and acetonitrile, or a mixed solvent of methanol, acetonitrile, and isopropanol;

[0023] Gradient elution or isocratic elution is used for sample separation.

[0024] The high-performance liquid system of the present invention employs a general-purpose high-pressure pump and an injector. Preferably, in the method of the present invention, Dikma Diamonsil C18 (150mm×2.1, 5μm) is used to separate plasma samples, and Ultimate XB C18 (50mm×2.1, 5μm) is used to separate cerebrospinal fluid samples.

[0025] Furthermore, when the mobile phase A is an aqueous solution containing ammonium formate, the concentration of the aqueous solution containing ammonium formate is 5 - 20 mM;

[0026] When the mobile phase B is a mixed solvent of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 10:90 - 90:10

[0027] As a preferred embodiment, the concentration of the aqueous solution containing ammonium formate is 10 mM;

[0028] The volume ratio of methanol to acetonitrile is 50:50.

[0029] Furthermore, the gradient elution program for the plasma sample to be measured is as follows: 0 - 0.5 min, 10% B phase; 0.5 - 2.0 min, 10 - 20% B phase; 2.0 - 3.5 min, 20 - 60% B phase; 3.5 - 5.0 min, 60 - 85% B phase; 5.0 - 6.5 min, 85 - 88% B phase; 6.5 - 6.6 min, 88 - 95% B phase; 6.6 - 7.50 min, 95% B phase; 7.50 - 7.51 min, 95 - 10% B phase; 7.51 - 8.50 min, 10% B phase;

[0030] Alternatively, the gradient elution program for the cerebrospinal fluid sample to be measured is as follows: 0 - 0.5 min, 30% B phase; 0.5 - 1.0 min, 30 - 65% B phase; 1.0 - 2.5 min, 65 - 80% B phase; 2.5 - 2.6 min, 80 - 95% B phase; 2.6 - 3.4 min, 95% B phase; 3.40 - 3.41 min, 95 - 30% B phase; 3.41 - 4.0 min, 30% B phase.

[0031] Furthermore, the mass spectrometry conditions of the LC-MS / MS instrument include: electrospray ionization or atmospheric pressure chemical ionization; multiple reaction monitoring (MRM) positive ion mode; ion channels: m / z→533.4 / 433.2 (olverembatinib) and m / z→502.4 / 394.2 (internal standard imatinib d8).

[0032] The present invention also provides a kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid, which kit comprises olverembatinib standard curve working solutions of different concentrations, olverembatinib quality control working solutions of different concentrations and an internal standard working solution.

[0033] The present invention uses a universal tandem mass detector to detect the peak areas of olverembatinib and the internal standard in a sample; preferably, the mass spectrometry parameters of this method are set as follows: curtain gas (CUR) 30 psi; collision-activated dissociation (CAD) 6; electrospray voltage 5500 V; ion source temperature 500; ion source gases 1 and 2 are 40 psi and 50 psi. The collision energy of olverembatinib is 27.7 V and the declustering voltage is 100 V; the collision energy of the internal standard is 36 V and the declustering voltage is 68 V.

[0034] Advantages of the present invention:

[0035] In the method for determining the concentration of olverembatinib in human plasma and cerebrospinal fluid according to the present invention, after simple pretreatment of the sample to be tested, it is detected by an LC-MS / MS instrument. Olverembatinib has a good linear relationship in the range of 500 - 50000 pg / mL in plasma samples and 10 - 1000 pg / ml in cerebrospinal fluid samples; the method has a high recovery rate, good stability, and the matrix does not affect the detection of the analyte and the internal standard, and the precision and accuracy within and between batches both meet the standards.

[0036] The present invention not only has simple pretreatment, low cost, short analysis time, and good specificity, but also has high sensitivity of the detection method, and the lower limit of quantification can reach the picogram level, which is rare in other LC-MS / MS detection methods. This method can be used for the pharmacokinetic-pharmacodynamic research of olverembatinib and the determination of the concentration of olverembatinib in the plasma and cerebrospinal fluid of routine clinical patients. Description of the Drawings

[0037] Figure 1 For the molecular structure and secondary mass spectrum of olverembatinib (A) and the internal standard imatinib d8 (B);

[0038] In the figure, A is the molecular structure and secondary mass spectrum of olverembatinib;

[0039] B is the molecular structure and secondary mass spectrum of the internal standard imatinib d8;

[0040] Figure 2 For the characteristic chromatograms of blank plasma samples, blank plasma samples containing the internal standard, and LLOQ samples;

[0041] In the figure, A is the characteristic chromatogram of a blank plasma sample;

[0042] B is the characteristic chromatogram of a blank plasma sample containing the internal standard;

[0043] C is the characteristic chromatogram of the LLOQ sample;

[0044] Figure 3 are the characteristic chromatograms of the blank cerebrospinal fluid sample, the artificial cerebrospinal fluid sample containing the internal standard, and the LLOQ sample;

[0045] In the figure, A is the characteristic chromatogram of the blank cerebrospinal fluid sample;

[0046] B is the characteristic chromatogram of the artificial cerebrospinal fluid sample containing the internal standard;

[0047] C is the characteristic chromatogram of the LLOQ sample. Detailed implementation mode

[0048] The following further describes the present invention in detail with reference to specific embodiments for those skilled in the art to understand.

[0049] The method for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS of the present invention includes the following steps:

[0050] 1) Prepare olverembatinib standard curve working solutions with different concentrations and olverembatinib quality control working solutions with different concentrations;

[0051] 2) Prepare the internal standard working solution;

[0052] 3) Add the internal standard working solution to the sample to be tested, vortex mix, then perform pretreatment to obtain the supernatant, and inject the supernatant into the LC-MS / MS instrument for detection and analysis.

[0053] In the method of the present invention, the following preferred conditions can be considered. The methods included are the selection of liquid phase conditions, chromatographic conditions, and sample pretreatment methods. Among them, for the liquid phase conditions, the chromatographic column selected for the detection of olverembatinib in plasma samples is Dikma Diamonsil C18 (150mm×2.1, 5μm), and the chromatographic column used for the determination of cerebrospinal fluid samples is Ultimate XB C18 (50mm×2.1, 5μm). The mobile phase used for the detection of both samples is: 10mM ammonium formate water and methanol-acetonitrile (v:v, 50:50). The flow rate for the detection of plasma samples is 0.60 mL / min, and the total flow rate for the detection of cerebrospinal fluid samples is 0.50 mL / min, and gradient elution is performed for both. Plasma samples are pretreated by liquid-liquid extraction with methyl tert-butyl ether, while cerebrospinal fluid samples are pretreated by protein precipitation with methanol. The mass spectrometry conditions are electrospray ionization, positive ion mode of multiple reaction ion monitoring, and the selected ion pairs for the analyte and internal standard are: m / z→533.4 / 433.2 (olverembatinib) and m / z→502.4 / 394.2 (internal standard imatinib d8).

[0054] 1. Materials and Methods:

[0055] 1.1 Instruments and Reagents:

[0056] LC-20AD high-performance liquid chromatography pump (Shimadzu; Japan), equipped with SIL-20AC HT automatic sampler and CTO-20AC column oven; AB Sciex API4000 triple quadrupole mass spectrometer (AB Sciex; USA), equipped with electrospray ionization source (ESI) and atmospheric pressure chemical ionization source (APCI); Multifuge X3R high-speed refrigerated centrifuge (Thermo Fisher Scientific; USA); MS105DU analytical balance (METTLER TOLEDO; USA); Vortex-2 GENIE G560E vortex mixer (Scientific Industries; USA); PURELAB flex ultrapure water machine (ELGA; UK); HN200 multi-functional nitrogen blower (Hanon Instruments; Jinan, China).

[0057] Orexin standard (Guangzhou Jiantu, 0125-RD-0044); Imatinib d8 standard (TRC Research, 5-NKM-63-5); Ammonium formate (Aladdin Reagent Co., Ltd.; Shanghai, China); Artificial cerebrospinal fluid (Feijing Phygene scientific reagent; Fuzhou, China); Chromatographic grade methanol, acetonitrile, isopropanol (German Simark company), chromatographic grade methyl tert-butyl ether (Ron reagent; Shanghai, China) and ultrapure water (prepared by PURELAB flex ultrapure water machine).

[0058] 2. LC-MS Conditions

[0059] Plasma liquid phase conditions: Dikma Diamonsil C18 (150mm×2.1, 5μm) chromatographic column, column temperature 40°C, total flow rate 0.60 mL / min. Mobile phase A (phase A) is: 10 mmol ammonium formate water, mobile phase B (phase B) is: methanol-acetonitrile (50:50, v:v), gradient elution program is: 0 - 0.5 min, 10% B phase; 0.5 - 2.0 min, 10 - 20% B phase; 2.0 - 3.5 min, 20 - 60% B phase; 3.5 - 5.0 min, 60 - 85% B phase; 5.0 - 6.5 min, 85 - 88% B phase; 6.5 - 6.6 min, 88 - 95% B phase; 6.6 - 7.50 min, 95% B phase; 7.50 - 7.51 min, 95 - 10% B phase; 7.51 - 8.50 min 10% B phase. Injection volume 20 μL.

[0060] Liquid phase conditions for cerebrospinal fluid: Ultimate XB C18 (50mm×2.1, 5μm) chromatographic column, column temperature 40°C, total flow rate 0.50 mL / min, mobile phase same as plasma, phase A: 10 mmol ammonium formate in water, phase B: methanol - acetonitrile (50:50), gradient elution program: 0 - 0.5 min, 30% B phase; 0.5 - 1.0 min, 30 - 65% B phase; 1.0 - 2.5 min, 65 - 80% B phase; 2.5 - 2.6 min, 80 - 95% B phase; 2.6 - 3.4 min, 95% B phase; 3.40 - 3.41 min, 95 - 30% B phase; 3.41 - 4.0 min 30% B phase. Injection volume is 30 μL.

[0061] Mass spectrometry conditions: Electrospray ionization; Multiple reaction monitoring (MRM) positive ion mode; Ion channels: m / z→533.4 / 433.2 (olverembatinib) and m / z→502.4 / 394.2 (internal standard imatinib d8); Curtain gas (CUR) 30 psi; Collision - activated dissociation (CAD) 6; Electrospray voltage 5500 V; Ion source temperature 500; Ion source gases 1 and 2 are 40 psi and 50 psi. The collision energy of olverembatinib is 27.7 V and the declustering voltage is 100 V; The collision energy of the internal standard is 36 V and the declustering voltage is 68 V.

[0062] 3. Preparation of solutions

[0063] Precisely weigh two portions of olverembatinib standard, dissolve and make up the volume with methanol to prepare a standard stock solution with a concentration of 2 mg / mL. Precisely weigh imatinib d8, dissolve and make up the volume with methanol to prepare an internal standard stock solution with a concentration of 0.4 mg / mL. The above stock solutions are all stored at -80°C.

[0064] Dilute the olverembatinib stock solution with methanol - water (75:25, v:v) to prepare plasma standard curve working solutions with concentrations of 10, 20, 50, 100, 200, 500, 800, 1000 ng / mL and quality control working solutions with concentrations of 30, 160 and 750 ng / mL. Dilute the olverembatinib stock solution with methanol - water (75:25, v:v) to prepare cerebrospinal fluid standard curve working solutions with concentrations of 200, 500, 1000, 2000, 5000, 10000, 16000, 20000 pg / mL and quality control working solutions with concentrations of 600, 4000 and 15000 pg / mL. In addition, dilute the internal standard stock solution with pure methanol to obtain internal standard working solutions with two concentrations of 20 ng / mL (plasma) and 70 pg / mL (cerebrospinal fluid). The above working solutions are all stored at -20°C for standby.

[0065] Artificial cerebrospinal fluid sample preparation: To better simulate human cerebrospinal fluid samples, according to the method in reference [J Neurosci Methods. 2020 Jul 15; 341: 108760], commercially available artificial cerebrospinal fluid and bovine serum albumin (BSA) were used to prepare an artificial cerebrospinal fluid sample containing 0.5% BSA in this method.

[0066] 4. Pretreatment of samples to be tested

[0067] Pretreatment process of plasma samples to be tested: Precisely pipette 100 μL of plasma samples to be tested, add 20 μL of internal standard working solution, vortex and mix well. Then add 1 mL of MTBE solution, vortex for 5 min, centrifuge at 14000 rpm at 4°C for 10 min. Pipette 800 μL of the upper-layer MTBE solution, dry it under nitrogen at room temperature, and then re-dissolve it with 100 μL of methanol-water (75:25, v:v), vortex and mix well. Centrifuge at 14000 rpm at 4°C for 5 min, and take 20 μL of the supernatant for LC-MS / MS analysis.

[0068] Pretreatment process of cerebrospinal fluid samples to be tested: Add 3 times the volume of methanol working solution containing internal standard (internal standard concentration 70 pg / ml) to the cerebrospinal fluid samples to be tested (≥400 μL), vortex for 5 min. Then precisely pipette 1.60 mL of the sample, centrifuge at 14000 rpm at 4°C for 10 min. Pipette 1.4 mL of the supernatant, dry it under nitrogen at room temperature, and then re-dissolve it with 160 μL of methanol-water (75:25, v:v), vortex and mix well. Centrifuge at 14000 rpm at 4°C for 5 min, and take 30 μL of the supernatant for LC-MS / MS analysis.

[0069] 5. Contents of methodological investigation

[0070] The established method was verified methodologically according to the "Guidelines for the Validation of Quantitative Analysis Methods for Biological Samples" in the Chinese Pharmacopoeia (2020 Edition) to ensure the accuracy, repeatability and stability of the detection. The verification contents include: method specificity, standard curve, precision and accuracy, matrix effect, extraction recovery rate, residue and stability.

[0071] 5.1 Method specificity

[0072] Take 6 blank plasma samples from healthy human subjects of different batches, and prepare blank samples, blank samples containing internal standard and lower limit of quantitation (LLOQ) plasma samples respectively. After the pretreatment of plasma samples, inject them for analysis to obtain spectra (characteristic spectra such as Figure 2 and Figure 3 ). Prepare 6 parallel blank samples, blank samples containing internal standard and lower limit of quantitation cerebrospinal fluid samples with artificial cerebrospinal fluid, inject them for analysis to obtain spectra (characteristic spectra such asFigure 2 and Figure 3 )

[0073] The results showed that the endogenous substances in plasma samples and cerebrospinal fluid samples did not interfere with the determination of olverembatinib and the internal standard. The area of the interfering peak at the elution time of olverembatinib was less than 20.0% of the area of the peak at the lower limit of quantification, and the area of the interfering peak at the elution time of the internal standard was less than 5.0% of the area of the internal standard peak. The results met the requirements.

[0074] 5.2 Standard Curve

[0075] The standard plasma samples and standard cerebrospinal fluid samples were prepared according to the ratio of working solution to matrix of 1:20. 20 μL of the plasma sample working solution was taken and added to 380 μL of blank plasma to prepare plasma standard curve samples with concentrations of 500, 1000, 2500, 5000, 10000, 25000, 40000, 50000 pg / mL and plasma quality control samples with concentrations of 1500, 8000, 37500 pg / mL. 20 μL of the cerebrospinal fluid working solution was taken and added to 380 μL of artificial cerebrospinal fluid to prepare cerebrospinal fluid standard curve samples with concentrations of 10, 25, 50, 100, 250, 500, 800, 1000 pg / mL and cerebrospinal fluid quality control samples with concentrations of 30, 200, 750 pg / mL. The standard curves of three batches were investigated respectively. Taking the ratio f of the peak areas of olverembatinib and the internal standard as the ordinate and the analyte concentration as the abscissa, weighted (W = 1 / x 2 ) least squares method was used for regression calculation. The results showed that olverembatinib had good linearity in the ranges of 500 - 50000 pg / mL in plasma and 10 - 1000 pg / mL in cerebrospinal fluid, and the correlation coefficient r was greater than 0.99.

[0076] 5.3 Precision and Accuracy

[0077] Plasma and cerebrospinal fluid samples with LLOQ, low, medium, and high concentrations were prepared, and 5 replicates were prepared for each concentration. After sample pretreatment, they were injected for detection as a batch of precision and accuracy samples. Within more than two days, different three batches of precision and accuracy samples were prepared and determined, and the chromatograms were recorded. The precision and accuracy within and between batches were investigated by the relative standard deviation (RSD%) and accuracy bias (Bias%) of the measured concentrations. The results showed that the RSD% and Bias% of olverembatinib at LLOQ and 3 quality control concentrations within and between batches were all within 15%. The accuracy and precision of the method were good and met the requirements of the guiding principles (see Table 1 for details).

[0078] Table 1 Precision and Accuracy of Olverembatinib in Plasma and Cerebrospinal Fluid Samples

[0079]

[0080] 5.4 Matrix effect

[0081] Preparation of control group samples: Pipette 100.0 μL of pure water, add methanol solution with the same volume as the internal standard working solution, perform the plasma pretreatment steps, and finally add a mixed working solution with an olverembatin concentration of 1071.4 pg / mL and 26785.7 pg / mL and an internal standard concentration of 2.86 ng / mL during the reconstitution step. Prepare 3 samples in parallel for each concentration to obtain plasma control group samples; Pipette 400.0 μL of pure water, add methanol with the same volume as the internal standard working solution, perform the cerebrospinal fluid pretreatment steps, and finally add a mixed working solution with an olverembatin concentration of 65.6 pg / mL and 1640.6 pg / mL and an internal standard concentration of 459.4 pg / mL during the reconstitution step. Prepare 3 samples in parallel for each concentration to obtain cerebrospinal fluid control group samples.

[0082] Preparation of experimental group samples: Pipette 100 μL of blank plasma from 6 different healthy subjects, add methanol with the same volume as the internal standard working solution, perform the plasma pretreatment steps, and finally reconstitute according to the same steps as the preparation of control group samples to obtain plasma experimental group samples. Pipette 6 portions of 400 μL of artificial cerebrospinal fluid samples, add methanol with the same volume as the internal standard working solution, perform the cerebrospinal fluid pretreatment steps, and finally reconstitute according to the same steps as the preparation of control group samples to obtain cerebrospinal fluid experimental group samples.

[0083] Inject the above samples for analysis, and record the average peak area As of the analyte in the control group & and the peak area As of the analyte in the experimental group, the average peak area Ai of the internal standard in the control group & and the peak area Ai of the internal standard in the experimental group. Calculate the relative matrix effect of the analyte and the internal standard and the absolute matrix effect of the analyte according to the following formulas respectively:

[0084] Analyte absolute matrix effect ME = As / As & ×100%,

[0085] Internal standard absolute matrix effect ME = Ai / Ai & ×100%,

[0086] Relative matrix effect MF = Analyte ME / Internal standard ME × 100%. The results show that the RSD% of the normalized relative matrix effect of low and high concentration quality control samples of olverembatin in plasma and cerebrospinal fluid are both less than 10.0% (see Table 2 for details), and the matrix effect meets the requirements of the guiding principles.

[0087] Table 2 Matrix effect of olverembatin and internal standard

[0088]

[0089] 5.5 Extraction recovery rate

[0090] Control group samples: The blank plasma was pretreated for samples, and in the final reconstitution step, a mixed working solution with the concentration of olverembatin at 1071.4 pg / mL, 5714.3 pg / mL, 26785.7 pg / mL and the internal standard concentration at 2.86 ng / mL was added. Three samples were prepared in parallel for each concentration to obtain plasma control group samples; The blank artificial cerebrospinal fluid was pretreated for samples, and in the final reconstitution step, a mixed working solution with the concentration of olverembatin at 65.6 pg / mL, 437.5 pg / mL and 1640.6 pg / mL and the internal standard concentration at 459.4 pg / mL was added. Three samples were prepared in parallel for each concentration to obtain cerebrospinal fluid control group samples. Inject for analysis and calculate the average peak area As of olverembatin and the average peak area Bs of the internal standard at each concentration.

[0091] Experimental group samples: Low, medium and high concentration quality control samples of plasma and cerebrospinal fluid were prepared respectively. Five samples were prepared for each concentration. After sample pretreatment, inject for analysis and record the peak area Ai of olverembatin and the peak area Bi of the internal standard at each concentration. Substitute the above Ai, Bi, As and Bs into the following formula to obtain the extraction recovery rate (R%) of olverembatin:

[0092] Extraction recovery rate R% = Ai / As × 100% or R% = Bi / Bs × 100%

[0093] The results showed that the coefficient of variation of the extraction recovery rates of olverembatin at low, medium and high quality control concentrations in plasma and cerebrospinal fluid was ≤ 15.0%. The extraction recovery rates were uniform and good, and the results met the requirements.

[0094] Table 3 Extraction recovery rate of olverembatin

[0095]

[0096] 5.6 Residue

[0097] Prepare plasma and cerebrospinal fluid LLOQ, upper limit of quantification and blank samples, inject for analysis of the upper limit of quantification, blank samples and LLOQ samples in turn, and repeat 3 times to obtain the corresponding peak areas. The results showed that the residue of olverembatin was less than 20.0% of LLOQ, and the residue of the internal standard was less than 5.0%. The residues of olverembatin and the internal standard did not affect the accuracy of the determination results.

[0098] 5.7 Stability

[0099] Prepare low and high quality control samples of cerebrospinal fluid and plasma separately, subject them to three cycles of freezing and thawing at -80°C and room temperature, leave them at room temperature for 24 hours, and store them in a -20°C refrigerator for 30 days. After sample pretreatment, quantify them using a freshly prepared standard curve to investigate the freeze-thaw, room temperature, and long-term stability of the samples. Prepare low and high quality control samples separately, subject them to sample pretreatment, and then store them in a 4°C refrigerator for 24 hours. After 24 hours, quantify them using a freshly prepared standard curve to investigate the stability of the treated samples. The results show that olverembatinib has good stability under the above conditions.

[0100] Example 1

[0101] 1. A kit for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid, the kit comprising olverembatinib standard curve working solutions of different concentrations, olverembatinib quality control working solutions of different concentrations, and internal standard working solutions; wherein, the internal standard working solutions are divided into clozapine working solution, deuterated imatinib working solution, and deuterated olverembatinib working solution.

[0102] 2. A method for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid using the above kit and based on LC-MS / MS, comprising the following steps:

[0103] 1) Prepare olverembatinib standard curve working solutions of different concentrations and olverembatinib quality control working solutions of different concentrations;

[0104] 2) Prepare internal standard working solutions;

[0105] 3) Add the internal standard working solutions to the sample to be tested, vortex mix, then perform pretreatment to obtain a supernatant, and inject the supernatant into an LC-MS / MS instrument for detection and analysis.

[0106] 3. Use the above kit and method to determine clinical samples

[0107] Seven CML patients taking olverembatinib were enrolled for determination of the blood drug concentration or cerebrospinal fluid concentration of olverembatinib. Clozapine, deuterated imatinib, and deuterated olverembatinib were used as internal standards for quantification respectively. It can be seen from Table 4 that there is no significant difference in the quantified concentrations using the three internal standards. The olverembatinib concentration range in the plasma of the patients was 1.72 ng / mL to 25.74 ng / mL, and the cerebrospinal fluid concentration range was 137.9 pg / mL to 455.6 pg / mL, both within the linear range of the plasma and cerebrospinal fluid determination methods (see Table 4 for details).

[0108] Table 4 Plasma and Cerebrospinal Fluid Concentrations of Patients Taking Olverembatinib

[0109]

[0110] In summary: The present invention conducted a methodological investigation in accordance with the "Guiding Principles for the Validation of Quantitative Analytical Methods for Biological Samples" in the Chinese Pharmacopoeia (2020 Edition), and used this method to determine the concentration of olverembatinib in the plasma and cerebrospinal fluid of patients. The results of the methodological validation showed that olverembatinib had good linearity in the ranges of 500 - 50,000 pg / mL in plasma and 10 - 1,000 pg / mL in cerebrospinal fluid (r > 0.99). The within-batch and between-batch precision RSD% of all quality control samples in plasma and cerebrospinal fluid were less than 15%, and the bias of accuracy was within ±15%. The extraction recovery rate of olverembatinib in plasma was greater than 60%, and the extraction recovery rate of olverembatinib in cerebrospinal fluid was greater than 90%. The relative matrix effect RSD% of plasma and cerebrospinal fluid samples normalized by the internal standard was less than 10.0%. Plasma and cerebrospinal fluid samples of 7 patients were detected using this method. The concentration range of plasma samples was 1.72 ng / mL - 25.74 ng / mL, and the concentration range of cerebrospinal fluid samples was 137.9 pg / mL - 455.6 pg / mL. The olverembatinib concentration detection method established by the present invention has high sensitivity, good repeatability, short analysis time, good specificity and stability. The detection kit can meet the requirements for the quantitative analysis of biological samples of drug concentrations in the plasma and cerebrospinal fluid of patients treated with olverembatinib.

[0111] Other parts not described in detail are prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the concentration of olverembatinib in human plasma or cerebrospinal fluid based on LC-MS / MS, characterized in that: It includes the following steps: 1) Prepare standard curve working solutions of olverembatinib at different concentrations and quality control working solutions of olverembatinib at different concentrations; 2) Prepare internal standard working solution; 3) Add the internal standard working solution to the sample to be tested, vortex mix, then perform pretreatment to obtain the supernatant, and inject the supernatant into an LC-MS / MS instrument for detection and analysis; wherein, the sample to be tested is a plasma sample to be tested or a cerebrospinal fluid sample to be tested; The pretreatment steps for the plasma sample to be tested are as follows: Add the internal standard working solution to the plasma sample to be tested, vortex mix, and then add methyl tert-butyl ether and vortex; then centrifuge at low temperature and high speed, take the upper organic solvent and dry it with nitrogen, then re-dissolve it with a reconstitution solution, vortex mix well, and centrifuge at low temperature and high speed to obtain the supernatant; wherein, the volume of methyl tert-butyl ether is 5 to 20 times that of the plasma sample to be tested; Alternatively, the pretreatment steps for the cerebrospinal fluid sample to be tested are as follows: Add the internal standard working solution to the cerebrospinal fluid sample to be tested, vortex mix, then centrifuge at low temperature and high speed, take the upper organic solvent and dry it with nitrogen, then re-dissolve it with a reconstitution solution, vortex mix well, and centrifuge at low temperature and high speed to obtain the supernatant, and the volume of the internal standard working solution is 2 to 10 times that of the cerebrospinal fluid sample to be tested; The chromatographic conditions of the LC-MS / MS instrument are as follows: Using a universal C18 silica column as the chromatographic column, When mobile phase A is an aqueous solution containing ammonium formate, the concentration of the aqueous solution containing ammonium formate is 5 - 20 mM; When the mobile phase B is a mixed solvent of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 10:90 - 90:10; Gradient elution or isocratic elution is used for sample separation; The gradient elution program for the plasma sample to be tested is: 0 - 0.5 min, 10% phase B; 0.5 - 2.0 min, 10 - 20% phase B; 2.0 - 3.5 min, 20 - 60% phase B; 3.5 - 5.0 min, 60 - 85% phase B; 5.0 - 6.5 min, 85 - 88% phase B; 6.5 - 6.6 min, 88 - 95% phase B; 6.6 - 7.50 min, 95% phase B; 7.50 - 7.51 min, 95 - 10% phase B; 7.51 - 8.50 min, 10% phase B; Alternatively, the gradient elution program for the cerebrospinal fluid sample to be tested is: 0 - 0.5 min, 30% phase B; 0.5 - 1.0 min, 30 - 65% phase B; 1.0 - 2.5 min, 65 - 80% phase B; 2.5 - 2.6 min, 80 - 95% phase B; 2.6 - 3.4 min, 95% phase B; 3.40 - 3.41 min, 95 - 30% phase B; 3.41 - 4.0 min, 30% phase B.

2. The method according to claim 1, wherein: In step 2), the internal standard is any one of clozapine, deuterated imatinib, and deuterated olverembatinib.

3. The method according to claim 1, characterized in that: The internal standard is deuterated imatinib d8; and when pretreating the plasma sample to be tested, the internal standard working solution is diluted with methanol, and the concentration is 20 ng / mL; Alternatively, the internal standard is deuterated imatinib d8; when pretreating the cerebrospinal fluid sample to be tested, the internal standard working solution is diluted with methanol, and the concentration is 70 pg / mL.

4. The method according to claim 1, characterized in that: The complex solution is prepared by mixing methanol or acetonitrile and water in a volume ratio of 50:50 to 90:10.

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