CN115656373A - Method and kit for determining concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry) - Google Patents

Abstract

The invention discloses a method and a kit for determining concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS; firstly, preparing working solution of an Olympic Lei Bati Ny standard curve with different concentrations and an Olympic Lei Bati Ny quality control working solution with different concentrations; then preparing internal standard working solution; and finally, adding the internal standard working solution into the sample to be detected, carrying out vortex mixing, carrying out pretreatment to obtain a supernatant, and injecting the supernatant into an LC-MS/MS instrument for detection and analysis. The invention has the advantages of simple pretreatment, low cost, short analysis time, good specificity and high sensitivity of the detection method, and the lower limit of the quantification can reach the picogram level, which is rare in other LC-MS/MS detection methods. The method can be used for pharmacokinetic-pharmacodynamic research of the Olympic Lei Bati Ni and the concentration determination of the Olympic Lei Bati Ni in the plasma and cerebrospinal fluid of a conventional clinical patient.

Description

Method and kit for determining concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry)

Technical Field

The invention relates to the field of drug concentration detection and analysis, in particular to a method and a kit for determining concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS.

Background

The O Lei Bati Ni (trade name: resistant rick, english name: olvembatinib) is a new medicine originally researched in China, is listed on the market in 11 months in 2021, is an oral third-generation tyrosine kinase BCR-ABL inhibitor, and is the first third-generation BCR-ABL targeted drug-resistant chronic myelocytic leukemia treatment medicine listed on the market in China. Drug resistance during the treatment of the current generation (imatinib) and the second generation (nilotinib, dasatinib and the like) tyrosine kinases is a common problem, and the T315I mutation is the leading cause of drug resistance. The Lei Bati ni has outstanding effect on various BCR-ABL mutants including the T315I mutation.

Because of the near marketing of the Lei Bati ni, the pharmacokinetic-pharmacodynamic condition in human body, especially in patients, is not clear and the clinical application is limited. The detection of the drug concentration in blood or other biological samples is the basis of pharmacokinetics, pharmacodynamics research and clinical accurate treatment, and no literature and patent report about the detection of the concentration of the ao Lei Bati ni exists at home and abroad at present, which hinders the research of drug treatment related to the ao Lei Bati ni.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method and a kit for determining the concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS, and the method has the advantages of rapidness, simplicity, convenience, sensitivity, high recovery rate, small matrix effect and the like; the method does not need expensive reagents, is simple to operate, and can quickly, sensitively and stably determine the concentration of the Lei Bati ni in plasma and cerebrospinal fluid; provides a foundation for the pharmacokinetics, pharmacodynamics and precise treatment research of the Olympic Lei Bati Nib.

In order to achieve the purpose, the invention designs a method for determining the concentration of the ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS, which comprises the following steps:

1) Preparing working solution of an Olympic Lei Bati Ny standard curve with different concentrations and an Olympic Lei Bati Ny quality control working solution with different concentrations;

2) Preparing an internal standard working solution;

3) Adding internal standard working solution into a sample to be detected, carrying out vortex mixing, carrying out pretreatment to obtain supernatant, and injecting the supernatant into an LC-MS/MS instrument for detection and analysis.

Further, in the step 2), the internal standard is any one of clozapine, deuterated imatinib and deuterated ao Lei Bati ni.

Still further, the sample to be detected is a plasma sample to be detected or a cerebrospinal fluid sample to be detected.

Still further, the pretreatment step of the plasma sample to be detected comprises the following steps:

adding an internal standard working solution into a plasma sample to be detected, carrying out vortex mixing, and then adding methyl tert-butyl ether (MTBE) for vortex; then centrifuging at a high speed at a low temperature, taking an upper layer organic solvent, drying by using nitrogen, redissolving by using a complex solution, uniformly mixing by vortex, and centrifuging at a high speed at a low temperature to obtain a supernatant; wherein the volume of the methyl tert-butyl ether (MTBE) is 5 to 20 times of that of the plasma sample to be detected;

or the pretreatment step of the cerebrospinal fluid sample to be detected comprises the following steps:

adding internal standard working solution into the cerebrospinal fluid sample to be detected, carrying out vortex mixing, then carrying out low-temperature high-speed centrifugation, taking an upper layer organic solvent nitrogen for drying, then re-dissolving by using a complex solution, carrying out vortex mixing, carrying out low-temperature high-speed centrifugation to obtain supernatant, wherein the volume of the internal standard working solution is 2-10 times that of the cerebrospinal fluid sample to be detected.

Still further, the internal standard is imatinib deuterated d8; when the plasma sample to be detected is pretreated, the internal standard working solution is diluted by methanol, and the concentration is 20ng/mL;

or the internal standard is imatinib deuterated d8; when the cerebrospinal fluid sample to be detected is pretreated, the internal standard working solution is diluted by methanol, and the concentration is 70pg/mL.

Still further, the complex solution is prepared by mixing methanol or acetonitrile and water according to a volume ratio of 50-90.

Preferably, the complex solution is formed by mixing methanol or acetonitrile with water according to a volume ratio of 75.

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

a general C18 silica gel column is taken as a chromatographic column,

the mobile phase A is aqueous solution containing ammonium formate or aqueous solution containing ammonium acetate or ammonia water with the concentration of 0.5 percent;

the mobile phase B is methanol, acetonitrile, a mixed solvent of methanol and acetonitrile or a mixed solvent of methanol, acetonitrile and isopropanol;

and (3) separating the sample by adopting a gradient elution method or an isocratic elution method.

The high-efficiency liquid phase system adopts a general high-pressure pump and a sample injector. Preferably, the method of the invention uses Dikma Diamond C18 (150 mm X2.1,5 μm) to separate plasma samples and Ultimate XB C18 (50 mm X2.1,5 μm) to separate cerebrospinal fluid samples.

Still further, when the mobile phase A is an aqueous solution containing ammonium formate, the concentration of the aqueous solution containing ammonium formate is 5-20mM;

when the mobile phase B is a mixed solvent of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile is 10

Preferably, the concentration of the aqueous solution comprising ammonium formate is 10mM;

the volume ratio of the methanol to the acetonitrile is 50.

Still further, the gradient elution procedure of the plasma sample to be tested is as follows: 0-0.5min, 10% of phase B; 0.5-2.0 min, 10-20% of phase B; 2.0-3.5min, 20-60% of phase B; 3.5-5.0 min, 60-85% of phase B; 5.0-6.5min, 85-88% of phase B; 6.5-6.6min, 88-95% of phase B; 6.6-7.50min, 95% of phase B; 7.50-7.51min, 95-10% of phase B; 7.51-8.50min 10% of phase B;

or, the gradient elution procedure of the cerebrospinal fluid sample to be detected is as follows: 0 to 0.5min,30% of phase B; 0.5-1.0min, 30-65% of phase B; 1.0-2.5min, 65-80% of phase B; 2.5-2.6min, 80-95% of phase B; 2.6-3.4min, 95% of phase B; 3.40-3.41min, 95-30% of phase B; 30% of phase B at 3.41-4.0 min.

Still further, the mass spectrum conditions of the LC-MS/MS instrument comprise: electrospray ionization or atmospheric pressure chemical ionization; multiple Reaction Monitoring (MRM) positive ion mode; ion channel: m/z → 533.4/433.2 (Lei Bati ni) and m/z → 502.4/394.2 (internal standard imatinib d 8).

The invention also provides a kit for determining the concentration of Lei Bati ni in human plasma or cerebrospinal fluid, which comprises working solutions of an O Lei Bati ni standard curve with different concentrations, an O Lei Bati ni quality control working solution with different concentrations and an internal standard working solution.

The method adopts a universal tandem mass spectrometry detector to detect the peak areas of the O Lei Bati Ni and the internal standard in the sample; preferably, the mass spectrum parameters of the method are set as follows: air curtain air (CUR) 30psi; collisional Activated Dissociation (CAD) 6; the electrospray voltage is 5500V; an ion source temperature of 500; the ion source gases 1 and 2 are 40psi and 50psi. The collision energy of O Lei Bati is 27.7V, and the cluster removing voltage is 100V; the internal standard collision energy was 36V and the declustering voltage was 68V.

The invention has the beneficial effects that:

in the method for determining the concentration of the ox Lei Bati ni in human plasma and cerebrospinal fluid, a sample to be detected is subjected to simple pretreatment and then is detected by an LC-MS/MS instrument. The linear relationship between the O Lei Bati Ni in the range of 500-50000pg/mL in the plasma sample and 10-1000pg/mL in the cerebrospinal fluid sample is good; the method has high recovery rate and good stability, the matrix does not influence the detection of the object to be detected and the internal standard, and the precision and the accuracy between batches meet the standard.

The invention has the advantages of simple pretreatment, low cost, short analysis time, good specificity and high sensitivity of the detection method, and the lower limit of the quantification can reach the picogram level, which is rare in other LC-MS/MS detection methods. The method can be used for pharmacokinetic-pharmacodynamic research of the Olympic Lei Bati Ni and the concentration determination of the Olympic Lei Bati Ni in the plasma and cerebrospinal fluid of a conventional clinical patient.

Drawings

FIG. 1 is a molecular structure and secondary mass spectrum of Olympic Lei Bati Ni (A) and internal standard imatinib d8 (B);

in the figure, A is the molecular structure and secondary mass spectrum of On Lei Bati Ni;

b, internally labeling a molecular structure and a secondary mass spectrogram of imatinib d8;

FIG. 2 is a characteristic chromatogram of a blank plasma sample, a blank plasma sample containing an internal standard, and a LLOQ sample;

in the figure, A is a characteristic chromatogram of a blank plasma sample;

b is a characteristic chromatogram of a blank plasma sample containing an internal standard;

c is a characteristic chromatogram of the LLOQ sample;

FIG. 3 is a characteristic chromatogram of a blank cerebrospinal fluid sample, an artificial cerebrospinal fluid sample containing an internal standard, and an LLOQ sample;

in the figure, A is a characteristic chromatogram of a blank cerebrospinal fluid sample;

b is a characteristic chromatogram of an artificial cerebrospinal fluid sample containing an internal standard;

c is a characteristic chromatogram of the LLOQ sample.

Detailed Description

The present invention is described in further detail below with reference to specific examples so that those skilled in the art can understand the invention.

The method for determining the concentration of the ox Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS comprises the following steps:

1) Preparing working solution of an Olympic Lei Bati Ny standard curve with different concentrations and an Olympic Lei Bati Ny quality control working solution with different concentrations;

2) Preparing an internal standard working solution;

3) Adding internal standard working solution into a sample to be detected, carrying out vortex mixing, carrying out pretreatment to obtain supernatant, and injecting the supernatant into an LC-MS/MS instrument for detection and analysis.

In the method of the present invention, the following preferable conditions can be considered. The method comprises the selection of liquid phase conditions, chromatographic conditions and sample pretreatment methods, wherein for the liquid phase conditions, a chromatographic column selected for detecting Oxa Lei Bati Ni in a plasma sample is Dikma Diamonsil C18 (150 mm multiplied by 2.1,5 μm), a chromatographic column used for detecting a cerebrospinal fluid sample is Ultimate XB C18 (50 mm multiplied by 2.1,5 μm), and mobile phases used for detecting the two samples are both: 10mM ammonium formate water and methanol-acetonitrile (v: v, 50), wherein the flow rate detected for plasma samples was 0.60mL/min and the total flow rate detected for cerebrospinal fluid samples was 0.50mL/min, were all subjected to gradient elution. The plasma samples are pretreated by a methyl tert-butyl ether liquid-liquid extraction mode, and the cerebrospinal fluid samples are pretreated by methanol protein precipitation. The mass spectrometry conditions are electrospray ionization and multi-reaction ion monitoring positive ion mode, and the ion pairs selected for the analyte and the internal standard are respectively as follows: m/z → 533.4/433.2 (Lei Bati ni) and m/z → 502.4/394.2 (internal standard imatinib d 8).

1. The material and the method are as follows:

1.1 Instrument and reagents:

LC-20AD high performance liquid chromatography pump (Shimadzu; japan) equipped with SIL-20ACHT autosampler and CTO-20AC column incubator; AB Sciex API4000 triple quadrupole mass spectrometer (AB Sciex; USA) equipped with electrospray ion source (ESI) and atmospheric pressure chemical ionization ion source (APCI); multifuge X3R type high speed refrigerated centrifuge (Thermo Fisher Scientific; USA); MS105DU analytical balance (METTLER TOLEDO; USA); vortex mixer Vortex-2 GENEE G560E (Scientific Industries; USA); PURELA flex model ultrapure water machine (ELGA; UK); HN200 multifunctional Nitrogen blowing apparatus (energy apparatus; china Jinan).

O Lei Bati Ny Standard (Guangzhou good-pass, 0125-RD-0044); imatinib d8 standard (TRC Research, 5-NKM-63-5); ammonium formate (Aladdin reagents, inc.; shanghai, china); artificial cerebrospinal fluid (feijing Phygene research reagent; fuzhou, china); methanol, acetonitrile, isopropanol (Simark, germany), methyl tert-butyl ether (Ron's reagent; shanghai, china) and ultrapure water (produced by PURELA flex water purifier) of chromatographic grade.

2. Liquid condition

Plasma liquid phase conditions: dikma Diamonsil C18 (150 mm. Times. 2.1,5 μm) column, column temperature 40 ℃ and total flow rate 0.60mL/min. Mobile phase a (phase a) was: 10mmol of ammonium formate water, mobile phase B (phase B) is: methanol-acetonitrile (50, v): 0-0.5min, 10% of phase B; 0.5-2.0 min, 10-20% of phase B; 2.0-3.5min, 20-60% of phase B; 3.5-5.0 min, 60-85% of phase B; 5.0-6.5min, 85-88% of phase B; 6.5-6.6min, 88-95% of phase B; 6.6 to 7.50min,95% of phase B; 7.50-7.51min, 95-10% of phase B; 7.51-8.50min 10% of phase B. The injection volume was 20. Mu.L.

Cerebrospinal fluid phase conditions: ultimate XB C18 (50 mm × 2.1,5 μm) column, column temperature 40 ℃, total flow rate 0.50mL/min, flow the same plasma, phase a: 10mmol of ammonium formate in water B phase: methanol acetonitrile (50: 0-0.5min, 30% of phase B; 0.5-1.0 min, 30-65% of phase B; 1.0-2.5min, 65-80% of phase B; 2.5-2.6min, 80-95% of phase B; 2.6-3.4min, 95% of phase B; 3.40-3.41min, 95-30% of phase B; 30% of B phase within 3.41-4.0min. The injection volume was 30. Mu.L.

Mass spectrum conditions: carrying out electrospray ionization; multiple Reaction Monitoring (MRM) positive ion mode; ion channel: m/z → 533.4/433.2 (Lei Bati ni) and m/z → 502.4/394.2 (internal standard imatinib d 8); air curtain air (CUR) 30psi; collisional Activated Dissociation (CAD) 6; the electrospray voltage is 5500V; an ion source temperature 500; the ion source gases 1 and 2 are 40psi and 50psi. The collision energy of O Lei Bati is 27.7V, and the cluster removing voltage is 100V; the internal standard collision energy was 36V and the declustering voltage was 68V.

3. Preparation of the solution

Two parts of the Olympic Lei Bati Ni standard substance are precisely weighed, dissolved by methanol and subjected to constant volume to prepare a standard substance stock solution with the concentration of 2 mg/mL. And accurately weighing imatinib d8, dissolving with methanol, diluting to constant volume, and preparing into an internal standard stock solution with the concentration of 0.4 mg/mL. The above stock solutions were stored at-80 ℃.

Stock solutions of ox Lei Bati ni were diluted with methanol-water (75, 25, v) and formulated as plasma standard curve working solutions at concentrations of 10, 20, 50, 100, 200, 500, 800, 1000ng/mL and quality control working solutions of 30, 160 and 750 ng/mL. The stock solution of ox Lei Bati ni was diluted with methanol-water (75, 25, v) to make cerebrospinal fluid standard curve working solutions with concentrations of 200, 500, 1000, 2000, 5000, 10000, 16000, 20000pg/mL and quality control working solutions of 600, 4000 and 15000 pg/mL. In addition, the internal standard stock was diluted with pure methanol to two concentrations of internal standard working solution of 20ng/mL (plasma) and 70pg/mL (cerebrospinal fluid). The working solutions are all stored at-20 ℃ for later use.

Preparing an artificial cerebrospinal fluid sample: to better simulate a human cerebrospinal fluid sample, the sample was tested according to the references [ J Neurosci methods.2020ju 15;341, 108760], prepared from commercially available artificial cerebrospinal fluid and Bovine Serum Albumin (BSA) to make an artificial cerebrospinal fluid sample containing 0.5% BSA.

4. Pretreatment of sample to be tested

Pretreatment process of plasma sample to be detected: precisely absorbing 100 mu L of a plasma sample to be detected, adding 20 mu L of an internal standard working solution, uniformly mixing by vortex, adding 1mL of MTBE solution, centrifuging for 10min at 4 ℃ 14000rpm after 5min by vortex, absorbing 800 mu L of upper MTBE solution, drying by nitrogen at normal temperature, re-dissolving by 100 mu L of methanol-water (75, 25, v).

Pretreatment of the cerebrospinal fluid sample to be detected: adding 3-time volume of methanol working solution containing an internal standard (the internal standard concentration is 70 pg/mL) into a cerebrospinal fluid sample (being equal to or more than 400 mu L) to be detected, after 5min of vortex, precisely sucking 1.60mL of sample, centrifuging for 10min at 4 ℃ and 14000rpm, sucking 1.4mL of supernatant solution, drying by nitrogen at normal temperature, then redissolving by 160 mu L of methanol-water (75, 25, v).

5. Methodology review content

The methodology verification is carried out on the established method according to the verification guiding principle of the quantitative analysis method of the biological samples in the 'Chinese pharmacopoeia' of 2020 edition, so as to ensure the accuracy, the repeatability and the stability of the detection. The verification content comprises the following steps: method specificity, standard curve, precision and accuracy, matrix effect, extraction recovery, residue and stability.

5.1 method specificity

Taking 6 blank plasma samples Of human healthy subjects from different batches Of sources, preparing a blank sample, a blank sample containing an internal standard and a Lower Limit Of Quantitation (LLOQ) plasma sample respectively, pretreating the blank sample by the plasma sample, and carrying out sample injection analysis to obtain a spectrogram (characteristic spectrograms are shown in figure 2 and figure 3). Preparing blank sample, blank sample containing internal standard and quantitative lower limit cerebrospinal fluid sample with artificial cerebrospinal fluid, preparing 6 parts in parallel, and analyzing by sample injection to obtain spectrogram (characteristic spectrogram is shown in figure 2 and figure 3).

The result shows that endogenous substances in the plasma sample and the cerebrospinal fluid sample do not interfere the measurement of the Olympic Lei Bati Ni and the internal standard, the interference peak area at the peak emergence time of the Olympic Lei Bati is less than 20.0 percent of the quantitative lower limit peak area, the interference peak area at the internal standard peak emergence time is less than 5.0 percent of the internal standard peak area, and the result meets the requirement.

5.2 Standard Curve

The standard plasma sample and the standard cerebrospinal fluid sample are prepared according to the proportion of working solution and matrix 1, 20 mu L of plasma sample working solution is sucked, 380 mu L of blank plasma is added, and plasma standard curve samples with the concentrations of 500, 1000, 2500, 5000, 10000, 25000, 40000 and 50000pg/mL and plasma quality control samples with the concentrations of 1500, 8000 and 37500pg/mL are prepared. 20 mu L of cerebrospinal fluid working solution is sucked, 380 mu L of artificial cerebrospinal fluid is added, and cerebrospinal fluid standard curve samples with the concentration of 10, 25, 50, 100, 250, 500g, 800 and 1000pg/mL and cerebrospinal fluid quality control samples with the concentration of 30, 200 and 750pg/mL are prepared. The standard curves of the three batches were examined separately, in order to obtain OrebaThe peak area ratio f of tinib to internal standard is plotted on the ordinate and the analyte concentration on the abscissa, using weighting (W = 1/x) ² ) The least square method performs regression operation. The results show that the O Lei Bati Ni has good linearity in the range of 500-50000pg/mL in plasma and 10-1000pg/mL in cerebrospinal fluid, and the correlation coefficients r are both larger than 0.99.

5.3 precision and accuracy

Preparing plasma and cerebrospinal fluid samples with low, medium and high concentrations of LLOQ, preparing 5 parts of each concentration in parallel, pretreating the samples, and performing sample injection detection to obtain a batch of samples with precision and accuracy. Three different batches of samples with precision and accuracy are prepared and measured in more than two days, chromatograms are recorded, and the precision and the accuracy between the batches are respectively inspected through measuring the relative standard deviation (RSD%) and the accuracy Bias (Bias%) of the concentration. The results show that the RSD% and the Bias% of the On Lei Bati NinLLOQ and 3 quality control concentration batches are within 15%, the accuracy and precision of the method are good, and the method meets the requirements of guiding principles (see Table 1 for details).

TABLE 1 precision and accuracy of Ox Lei Bati Nib plasma and cerebrospinal fluid samples

5.4 stromal Effect

Preparation of control group samples: sucking 100.0 mu L of pure water, adding a methanol solution with the same volume as that of the internal standard working solution, performing a plasma pretreatment step, finally adding a mixed working solution of O Lei Bati with the concentration of 1071.4pg/mL and 26785.7pg/mL and the internal standard concentration of 2.86ng/mL in a re-dissolving step, and preparing 3 samples in parallel for each concentration to obtain a plasma control group sample; sucking 400.0 mu L of pure water, adding methanol with the same volume as that of the internal standard working solution, performing a cerebrospinal fluid pretreatment step, finally adding a mixed working solution of the O Lei Bati Ni with the concentration of 65.6pg/mL, 1640.6pg/mL and the internal standard concentration of 459.4pg/mL in a redissolution step, and preparing 3 samples in parallel per concentration to obtain a cerebrospinal fluid control group sample.

Preparation of experimental group samples: absorbing 100 mu L of blank plasma of 6 different healthy subjects, adding methanol with the same volume as the internal standard working solution, performing a plasma pretreatment step, and finally re-dissolving the same sample preparation step of a control group to obtain a sample of a plasma experimental group. Sucking 6 parts of 400 mu L artificial cerebrospinal fluid sample, adding methanol with the same volume as the internal standard working solution, performing cerebrospinal fluid pretreatment, and finally re-dissolving the same as the control group sample preparation step to obtain the cerebrospinal fluid experimental group sample.

The sample is subjected to sample injection analysis, and the average value As of the peak areas of the analytes in the control group is recorded ^& The peak area As of the analyte in the experimental group and the average value Ai of the peak area of the internal standard in the control group ^& And the peak area Ai of the internal standard of the experimental group. The relative matrix effect of the analyte and internal standard and the absolute matrix effect of the analyte were calculated according to the following formulas, respectively:

absolute matrix effect of analyte ME = As/As ^& ×100％,

Internal standard absolute matrix effect ME = Ai/Ai ^& ×100％,

Relative matrix effect MF = analyte ME/internal standard ME × 100%, the results indicate that the oc Lei Bati ni in plasma and cerebrospinal fluid is low, and the normalized relative matrix effect RSD% of the high concentration quality control sample is less than 10.0% (see table 2 for details), and the matrix effect meets the requirement of the guiding principle.

TABLE 2 Olympic Lei Bati Ni and internal standard matric effect

5.5 extraction recovery

Control group samples: performing sample pretreatment on blank plasma, adding mixed working solution with the concentration of Olympic Lei Bati Ni of 1071.4pg/mL, 5714.3pg/mL and 26785.7pg/mL and the concentration of internal standard of 2.86ng/mL in the final redissolution step, and preparing 3 samples in parallel at each concentration to obtain a plasma control group sample; performing sample pretreatment on blank artificial cerebrospinal fluid, adding mixed working solutions of Lei Bati Ni with the concentrations of 65.6pg/mL, 437.5pg/mL and 1640.6pg/mL and an internal standard concentration of 459.4pg/mL in the final redissolution step, and preparing 3 samples in parallel for each concentration to obtain a cerebrospinal fluid control group sample. And (5) carrying out sample injection analysis, and calculating the average value As of the peak area of Lei Bati nylon and the average value Bs of the peak area of the internal standard of each concentration.

Experimental group samples: respectively preparing low, medium and high concentration quality control samples of plasma and cerebrospinal fluid, preprocessing each sample by 5 samples, then carrying out sample injection analysis, recording peak areas Ai and internal standard peak areas Bi of the concentrations of the Olympic acid Lei Bati Ni, and substituting the Ai and Bi, the As and the Bs into the following formula to obtain the extraction recovery rate (R%) of the Olympic acid Lei Bati Ni:

extraction recovery rate R% = Ai/As × 100% or R% = Bi/Bs × 100%

The results show that the variation coefficients of the extraction recovery rates of the ox Lei Bati ni in low, medium and high quality control concentrations in blood plasma and cerebrospinal fluid are all less than or equal to 15.0 percent, the extraction recovery rates are uniform and good, and the results meet the requirements.

TABLE 3 recovery of the ox Lei Bati ni extraction

5.6 residues

Preparing plasma and cerebrospinal fluid LLOQ, upper limit of quantitation and a blank sample, sequentially carrying out sample injection analysis on the upper limit of quantitation, the blank sample and the LLOQ sample, and repeating the steps for 3 times to obtain corresponding peak areas. The results show that the residues of Olympic Lei Bati Ni are less than 20.0% of LLOQ, the residues of the internal standard are less than 5.0%, and the residues of Olympic Lei Bati Ni and the internal standard do not influence the accuracy of the determination result.

5.7 stability

Respectively preparing low-quality and high-quality cerebrospinal fluid and plasma samples, repeatedly freezing and thawing at-80 ℃ and normal temperature for three times, standing at room temperature for 24h, standing at-20 ℃ for 30 days in a refrigerator, pretreating the samples, quantifying according to a freshly prepared standard curve, and inspecting the freezing and thawing, room temperature and long-term stability of the samples; respectively preparing low-quality control samples and high-quality control samples, pretreating the samples, putting the pretreated samples in a refrigerator at 4 ℃ for 24h, quantifying the samples according to a freshly prepared standard curve after 24h, and inspecting the stability of the treated samples. The results show that the O Lei Bati Ni is stable in the above conditions.

Example 1

1. The kit for determining the concentration of Lei Bati ni in human plasma or cerebrospinal fluid comprises working solutions of an O Lei Bati ni standard curve with different concentrations, an O Lei Bati ni quality control working solution with different concentrations and an internal standard working solution; wherein the internal standard working solution is divided into clozapine working solution, deuterated imatinib working solution and deuterated O Lei Bati Nib working solution.

2. The method for determining the concentration of the ox Lei Bati ni in human plasma or cerebrospinal fluid by using the kit and based on LC-MS/MS comprises the following steps:

1) Preparing working solution of an Olympic Lei Bati Ny standard curve with different concentrations and an Olympic Lei Bati Ny quality control working solution with different concentrations;

2) Preparing an internal standard working solution;

3) Adding internal standard working solution into a sample to be detected, carrying out vortex mixing, carrying out pretreatment to obtain supernatant, and injecting the supernatant into an LC-MS/MS instrument for detection and analysis.

3. Determination of clinical specimens using the above kits and methods

7 CML patients who took Lei Bati ni were selected and used for Lei Bati ni blood concentration or cerebrospinal fluid concentration determination, and clozapine, deuterated imatinib and deuterated Lei Bati ni were respectively used as internal standards for quantification, and the concentrations quantified by the three internal standards were not significantly different from each other as shown in Table 4. The ox Lei Bati ni concentration range for patient plasma is 1.72ng/mL to 25.74ng/mL and the cerebrospinal fluid concentration range is 137.9pg/mL to 455.6pg/mL, all within the linear range of plasma and cerebrospinal fluid assay methods (see table 4 for details).

TABLE 4 plasma and cerebrospinal fluid concentrations in patients taking Olympic Lei Bati Ni

In conclusion: the invention examines methodology according to 'biological sample quantitative analysis method verification guiding principle' in the 2020 edition Chinese pharmacopoeia, and uses the method to determine the concentration of the ao Lei Bati ni in the plasma and cerebrospinal fluid of a patient. The results of methodology validation show that O Lei Bati Ni is well linear (r > 0.99) in the range of 500-50000pg/mL plasma and 10-1000pg/mL cerebrospinal fluid. The precision RSD% of all quality control samples of plasma and cerebrospinal fluid in batches and between batches is less than 15%, and the accuracy is biased to be within +/-15%. The extraction recovery rate of the Ox Lei Bati Ni in the plasma is more than 60 percent, the extraction recovery rate of the Ox Lei Bati Ni in the cerebrospinal fluid is more than 90 percent, and the RSD percent of the plasma and cerebrospinal fluid samples is less than 10.0 percent through the internal standard normalization relative matrix effect. Plasma and cerebrospinal fluid samples of 7 patients were tested using this method, with plasma samples ranging in concentration from 1.72ng/mL to 25.74ng/mL and cerebrospinal fluid samples ranging in concentration from 137.9pg/mL to 455.6pg/mL. The method for detecting the concentration of the Ox Lei Bati Ni established by the invention has the advantages of high sensitivity, good repeatability, short analysis time, good specificity and stability, and the detection kit can meet the requirement of quantitative analysis of biological samples of the drug concentration in plasma and cerebrospinal fluid of an Ox Lei Bati Ni treatment patient.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims (10)

1. A method for determining the concentration of Lei Bati ni in human plasma or cerebrospinal fluid based on LC-MS/MS is characterized in that: the method comprises the following steps:

1) Preparing working solution of an Olympic Lei Bati Ny standard curve with different concentrations and an Olympic Lei Bati Ny quality control working solution with different concentrations;

2) Preparing an internal standard working solution;

3) Adding internal standard working solution into a sample to be detected, carrying out vortex mixing, carrying out pretreatment to obtain supernatant, and injecting the supernatant into an LC-MS/MS instrument for detection and analysis.

2. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid according to claim 1, wherein: in the step 2), the internal standard is any one of clozapine, deuterated imatinib and deuterated ozoit Lei Bati ni.

3. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 2, wherein: the sample to be detected is a plasma sample to be detected or a cerebrospinal fluid sample to be detected.

4. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 3, wherein: the pretreatment steps of the plasma sample to be detected are as follows:

adding an internal standard working solution into a plasma sample to be detected, carrying out vortex mixing, and adding methyl tert-butyl ether for vortex; then centrifuging at a high speed at a low temperature, taking an upper layer organic solvent, drying by using nitrogen, redissolving by using a complex solution, uniformly mixing by vortex, and centrifuging at a high speed at a low temperature to obtain a supernatant; wherein, the volume of the methyl tert-butyl ether is 5 to 20 times of that of the blood plasma sample to be detected;

or the pretreatment step of the cerebrospinal fluid sample to be detected comprises the following steps:

adding internal standard working solution into the cerebrospinal fluid sample to be detected, carrying out vortex mixing, then carrying out low-temperature high-speed centrifugation, taking an upper layer organic solvent nitrogen for drying, then re-dissolving by using a complex solution, carrying out vortex mixing, carrying out low-temperature high-speed centrifugation to obtain supernatant, wherein the volume of the internal standard working solution is 2-10 times that of the cerebrospinal fluid sample to be detected.

5. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 4, wherein: the internal standard is imatinib deuterated d8; when the plasma sample to be detected is pretreated, the internal standard working solution is diluted by methanol, and the concentration is 20ng/mL;

or, the internal standard is imatinib deuterated d8; when the cerebrospinal fluid sample to be detected is pretreated, the internal standard working solution is diluted by methanol, and the concentration is 70pg/mL.

6. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 4, wherein: the complex solution is prepared by mixing methanol or acetonitrile with water according to the volume ratio of 50-90.

7. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 1, wherein: the chromatographic conditions of the LC-MS/MS instrument are as follows:

a general C18 silica gel column is taken as a chromatographic column,

the mobile phase A is aqueous solution containing ammonium formate or aqueous solution containing ammonium acetate or ammonia water with the concentration of 0.5 percent;

the mobile phase B is methanol, acetonitrile, a mixed solvent of methanol and acetonitrile or a mixed solvent of methanol, acetonitrile and isopropanol;

and (3) separating the sample by adopting a gradient elution method or an isocratic elution method.

8. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 7, wherein: when the mobile phase A is an aqueous solution containing ammonium formate, the concentration of the aqueous solution containing ammonium formate is 5-20mM;

when the mobile phase B is a mixed solvent of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile is 10.

9. The LC-MS/MS-based method for determining the concentration of ox Lei Bati ni in human plasma and cerebrospinal fluid according to claim 7, wherein: the gradient elution procedure of the plasma sample to be tested is as follows: 0 to 0.5min,10% of phase B; 0.5-2.0min, and 10-20% of phase B; 2.0 to 3.5min,20 to 60 percent of phase B; 3.5-5.0min, 60-85% of phase B; 5.0 to 6.5min,85 to 88 percent of phase B; 6.5-6.6min, 88-95% of phase B; 6.6 to 7.50min,95% of phase B; 7.50-7.51min, 95-10% of phase B; 7.51-8.50min 10% of phase B;

or, the gradient elution procedure of the cerebrospinal fluid sample to be detected is as follows: 0 to 0.5min,30% of phase B; 0.5-1.0min, 30-65% of phase B; 1.0-2.5min, 65-80% of phase B; 2.5-2.6 min, 80-95% of phase B; 2.6-3.4 min,95% B phase; 3.40-3.41min, 95-30% of phase B; 3.41-4.0min 30% of phase B.

10. A kit for determining the concentration of ox Lei Bati ni in human plasma or cerebrospinal fluid, which is characterized in that: the kit comprises working solutions of an Olympic Lei Bati Ny standard curve with different concentrations, an Olympic Lei Bati Ny quality control working solution with different concentrations and an internal standard working solution.

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