Method for detecting plasma concentration of elacycline in serum

Abstract

The invention provides a method for detecting the blood concentration of elacycline in serum. The method comprises the following steps: pretreating a human serum sample; then detecting by adopting liquid chromatography-mass spectrometry, wherein a liquid chromatography condition mobile phase comprises ultrapure water (a mobile phase A) containing 0.1% of formic acid and acetonitrile (a mobile phase B); the temperature of the chromatographic column is kept at 45 DEG C, and the sample size is 2L; the flow velocity of the mobile phase is set to be 0.3 mL / min, and the total operation time is 4.5 minutes; in mass spectrum conditions, a positive electrospray ionization mode is adopted, multiple reaction monitoring (MRM) is adopted, and multi-reaction monitoring is adopted as a scanning mode. The method is high in sensitivity, the lowest quantitative concentration can reach 50 ng / mL, and accurate, rapid and sensitive determination of the elacycline in the human serum can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological quantitative detection, relates to the quantitative detection of the blood drug concentration of eravacycline, and specifically relates to a method for detecting the blood drug concentration of eravacycline in serum. Background Art

[0002] Eravacycline is a fluorine-substituted fully synthetic tetracycline antibacterial drug with a broad antibacterial spectrum and strong antibacterial activity. Its mechanism of action is to reversibly bind to the A site of the 30S subunit of the bacterial ribosome, intercept the binding site of aminoacyl-tRNA to the mRNA-ribosome complex, thereby inhibiting bacterial protein synthesis and resulting in the inhibition of bacterial growth. In the treatment of infectious diseases such as gram-positive, gram-negative bacteria (except Pseudomonas aeruginosa and Burkholderia), atypical bacterial pathogens, and Neisseria gonorrhoeae, eravacycline has become an important drug for the treatment of multi-drug resistant bacterial infections due to its broad antibacterial activity and good drug resistance.

[0003] In the process of clinical treatment, especially in the treatment of critically ill infected patients, individualized treatment with antibacterial drugs is necessary, which helps patients achieve the best therapeutic effect and at the same time curbs bacterial drug resistance. The dosage of antibacterial drugs should be adjusted according to the drug exposure and the sensitivity of the infection site. The pharmacokinetic (PK) and pharmacodynamic (PD) studies of antibacterial drugs are important bases for optimizing the use of antibacterial drugs. The PK / PD theory helps clinicians formulate more effective treatment plans, improve clinical efficacy, and reduce the generation of drug-resistant bacteria by comprehensively considering the dynamic changes of drugs in the body (PK) and the antibacterial effects of drugs on pathogenic bacteria (PD). PK parameters are affected by various pathophysiological factors or other iatrogenic factors, and it is difficult to accurately achieve the PK / PD target of antibacterial drugs. Blood drug concentration monitoring is particularly important for achieving clinical efficacy and ensuring medication safety.

[0004] Eravacycline was approved for marketing in China in March 2023. Currently, the research on the method for monitoring the blood drug concentration of eravacycline in the reports has certain limitations: (1) The linear range is small. The linear range of the standard curve established by the method is 1.0 ng / mL to 500.0 ng / mL, which cannot meet the detection of the peak concentration of eravacycline of about 2000 ng / mL. (2) In the method, the plasma protein precipitant is trifluoroacetic acid. Trifluoroacetic acid has a strong ion pair effect and is likely to interact with the hydrophobic bonding phase of the chromatographic column and the residual polar surface, resulting in difficulty in flushing the column clean, thereby shortening the service life of the column.

[0005] In summary, to ensure that the drug is within a safe and effective concentration range and to provide data resources for pharmacokinetic studies, developing a quantitative detection method for eravacycline with a wide quantitative detection range and high sensitivity is of great significance for improving its clinical therapeutic effect and ensuring patient safety. Summary of the Invention

[0006] Based on the above problems, the present invention aims to develop and validate an LC-MS / MS analysis method for quantitatively analyzing the concentration of eravacycline using serum as a sample, and apply this method to TDM research to provide technical support for clinical individualized treatment and PK / PD research.

[0007] The process of the method of the present invention is outlined as follows: First, perform pretreatment on human serum samples; then use liquid chromatography-mass spectrometry for detection: The mobile phase of the liquid chromatography conditions includes ultrapure water containing 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B); the column temperature is maintained at 45°C, the injection volume is 2 μL; the mobile phase flow rate is set at 0.3 mL / min, and the total running time is 4.5 minutes; in the mass spectrometry conditions, positive electrospray ionization mode is adopted, multiple reaction monitoring (MRM) is used, and the scanning mode is multi-reaction monitoring. This method has high sensitivity, and the lowest concentration that can be quantified can reach 50 ng / mL, enabling accurate, rapid, and sensitive determination of eravacycline in human serum.

[0008] The specific technical solution to be protected by the present invention is as follows:

[0009] The method for detecting the blood drug concentration of eravacycline in serum provided by the present invention includes the following steps:

[0010] A) Pretreatment of serum samples: Select 60% aqueous acetonitrile solution as a precipitant to remove impurities in the serum.

[0011] The specific pretreatment method is as follows: Place a certain amount of serum in a microcentrifuge tube, precisely pipette the same volume of internal standard solution, and pipette 8 times the volume of the serum of the aqueous acetonitrile solution. After vortexing for 1 min, centrifuge at a high speed of 14000 rpm / min for 5 min using a centrifuge, and then transfer a certain amount of supernatant to an injection vial for quantitative analysis by liquid chromatography-mass spectrometry; among them, the volume of the serum is 1 / 150 of the capacity of the microcentrifuge tube.

[0012] B) Use liquid chromatography-mass spectrometry to detect the content of eravacycline in the pretreated serum sample to be detected. The isotope of eravacycline (deuterated eravacycline) is used as an internal standard, and the content of eravacycline is calculated by the standard curve method.

[0013] Among them, the chromatographic conditions are as follows:

[0014] Chromatographic column model: Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 µm); column temperature was 45 °C, injection volume was 2 μL;

[0015] Mobile phase A: ultrapure water solution of 0.1% formic acid;

[0016] Mobile phase B: acetonitrile, purity was mass spectrometry grade;

[0017] Gradient elution was used to separate impurities, flow rate was 0.3 mL / min, total running time was 5.5 minutes, and the gradient elution process was as follows: 0 - 0.5 minutes, 1 - 30% B; 0.501 - 1.5 minutes, 30 - 98% B; 1.501 - 2.5 minutes, 98% B; 2.501 - 4.5 minutes, 1% B;

[0018] The needle washing solution was 50% methanol - water solution.

[0019] Mass spectrometry conditions were as follows: operating in positive electrospray ionization mode, spray voltage was 3500 V, ion transfer tube temperature was 350 °C, vaporization temperature was 400 °C, sheath gas flow rate was 50 L / min, auxiliary gas flow rate was 15 L / min, and scanning gas flow rate was 1 L / min;

[0020] MRM conditions for eravacycline: the m / z of the precursor ion was 559.2, the m / z of the product ion was 542.2, and the collision energy was 21 V; the MRM conditions for deuterated eravacycline were as follows: the m / z of the precursor ion was 567.21, the m / z of the product ion was 550.3, and the collision energy was 20 V.

[0021] Preferably, in step B), the standard curve included seven concentration points S1 - S7, and the method for establishing the standard curve was as follows: accurately weigh the eravacycline standard product into a 1 mL brown volumetric flask, dissolve it evenly with methanol and make up the volume to 1 mL to prepare an eravacycline stock solution with a concentration of 1 mg / mL for standby; dilute the eravacycline stock solution with 50% methanol / water to prepare standard curve working solutions with mass concentrations of 500, 1000, 2500, 5000, 10000, 30000, 50000 ng / mL.

[0022] Accurately pipette 270 μL of blank serum into a 1.5 mL EP tube, add 30 μL of the eravacycline standard curve working solution respectively, vortex and mix evenly to prepare 7 standard curve samples with mass concentrations of 50, 100, 250, 500, 1000, 3000, 5000 ng / mL. After processing the above seven standard addition samples according to the sample pretreatment method, inject them for determination. Using the peak area ratio of the compound to be measured to the internal standard as the Y - axis and the reference substance concentration as the X - axis, establish the standard curve.

[0023] The peak area ratio of ilarocycline measured in the serum sample to the peak area of the internal standard is substituted into the accompanying calibration curve to calculate the content of ilarocycline in the serum sample.

[0024] Furthermore, quality control solutions and internal standard working solutions are also required during the detection process. Among them, the preparation method of the quality control solution is as follows: Prepare an ilarocycline stock solution with a concentration of 1 mg / mL, and dilute the ilarocycline stock solution with 50% methanol / water into QC working solutions with mass concentrations of 2000, 20000, and 40000 mg / mL; use the QC working solutions to prepare a lower limit of quantification sample LLOQ with a mass concentration of 50 ng / mL and low-quality control samples QCL, medium-quality control samples QCM, and high-quality control samples QCH with mass concentrations of 200, 2000, and 4000 ng / mL;

[0025] The concentration of the internal standard working solution is 1500 ng / mL, and the preparation method is the same as that of the ilarocycline stock solution.

[0026] The beneficial guarantees and effects of the present invention are as follows:

[0027] (1) Wide detection range: Using serum as the sample, the quantitative range of the blood drug concentration of ilarocycline measured in the present invention is 50 - 5000 ng / mL. The quantitative range is wide, and the peak concentration of ilarocycline can be accurately detected, which can meet the needs of clinical monitoring of peak concentration and trough concentration;

[0028] (2) High sensitivity and good accuracy. The sensitivity of drug detection has been significantly improved by using the LC-MS / MS method, which can meet the clinical needs in the real world. This detection method has been verified and complies with the M10 guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) regarding the validation of bioanalytical methods and the analysis of study samples and the bioanalytical method regulations of the European Bioanalysis Forum (EBF).

[0029] (3) The detection method is stable and efficient. LC-MS / MS runs for 4.5 minutes, and this method has been verified and has good stability. Description of the Drawings

[0030] Figure 1 Shows the typical chromatograms of ilarocycline and internal standard ilarocycline-d in human serum determined by LC-MS / MS method: on the left is the chromatogram of ilarocycline; on the right is the chromatogram of the internal standard deuterated ilarocycline: (a) double blank serum sample, (b) blank serum sample (only containing internal standard), (c) lower limit of quantification serum sample, (d) clinical serum sample.

[0031] Figure 2 Shows the linearity and lower limit of quantification of ilarocycline detection. Detailed Implementation Modes

[0032] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods being well known to those of ordinary skill in the art and having been described in many publications.

[0033] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation manners are for illustrative purposes only.

[0034] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise noted, the reagents used in the examples of the present invention can be obtained from sales companies.

[0035] I. Preparation of working solution

[0036] 1.1 Preparation of stock solution and working solution

[0037] Accurately weigh the eravacycline standard in a 1 mL brown volumetric flask, dissolve it uniformly with methanol and make up to 1 mL to prepare an eravacycline stock solution with a concentration of 1 mg / mL for standby. Dilute the eravacycline stock solution with 50% methanol / water to prepare standard curve working solutions with mass concentrations of 500, 1000, 2500, 5000, 10000, 30000, 50000 ng / mL.

[0038] Prepare an eravacycline stock solution with a concentration of 1 mg / mL in the same way for the preparation of quality control (QC) working solutions. Dilute the eravacycline stock solution with 50% methanol / water to prepare QC working solutions with mass concentrations of 2000, 20000, 40000 mg / mL.

[0039] Prepare a 1500 ng / mL internal standard working solution (deuterated eravacycline) in the same way. The stock solution and working solution are both placed in a -80 °C refrigerator for standby.

[0040] 1.2 Preparation of standard curve samples and quality control samples

[0041] Accurately pipette 270 μL of blank serum into a 1.5 mL EP tube, and add 30 μL of the standard curve working solution of eravacycline respectively. Vortex and mix well to prepare 7 standard curve samples with mass concentrations ranging from 50 to 5000 ng / mL. Using the same method, prepare a lower limit of quantification (LLOQ) sample with a mass concentration of 50 ng / mL and low-quality control samples (QCL), medium-quality control samples (QCM), and high-quality control samples (QCH) with mass concentrations of 200, 2000, and 4000 ng / mL using the QCL working solution. Among them, the LLOQ and 3 concentration levels of quality control samples are used for the verification of the lower limit of quantification and the accuracy and precision of the method in subsequent method verification, etc. The prepared serum samples are stored in a -80°C refrigerator for later use.

[0042] 1.3 Eluent Preparation

[0043] It includes eluent A and eluent B, which are used as mobile phase A and mobile phase B for ultra-high performance liquid chromatography respectively.

[0044] Mobile phase A is an ultrapure aqueous solution of 0.1% formic acid. Accurately pipette 0.5 mL of formic acid, dissolve it in ultrapure water, and make the volume up to 500 mL to obtain it; Mobile phase B: acetonitrile, with a purity of mass spectrometry grade.

[0045] II. Detection of Eravacycline Blood Concentration in Serum

[0046] 2.1 Serum Sample Pretreatment

[0047] The serum samples are from actual clinical TDM samples.

[0048] The protein precipitation method is used for serum sample pretreatment. The precipitant is 60% acetonitrile water, which can remove the protein components in the serum and avoid the contamination of the mass spectrometer by impurities in the serum, thereby inhibiting the detection of the target analyte. The specific sample treatment operation steps are as follows: Place 20 μL of serum in a 1.5 mL microcentrifuge tube, accurately pipette 20 μL of the internal standard solution, pipette 160 μL of acetonitrile water, vortex for 1 min, centrifuge at a high speed of 14000 rpm / min for 5 min using a centrifuge, and then transfer 100 μL of the supernatant to an injection vial for quantitative analysis by liquid chromatography-mass spectrometry.

[0049] 2.2 Liquid Chromatography-Mass Spectrometry Detection

[0050] LC-MS / MS analysis was performed on a Thermo UltiMate 3000 ultra-high performance liquid chromatograph and a TSQ ENDURA triple quadrupole mass spectrometer. The content of eravacycline in the pretreated serum samples to be tested was used, and the isotope of eravacycline (deuterated eravacycline) was used as the internal standard, and the content of eravacycline was calculated by the standard curve method.

[0051] 2.2.1 Chromatographic conditions

[0052] Column model: Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 µm); column temperature was 45 °C, injection volume was 2 μL;

[0053] The needle wash solution was 50% methanol-aqueous solution.

[0054] Mobile phase A: ultra-pure aqueous solution of 0.1% formic acid;

[0055] Mobile phase B: acetonitrile, with a purity of mass spectrometry grade;

[0056] Gradient elution was used to separate impurities, the flow rate was 0.3 mL / min, and the total running time was 4.5 minutes. The gradient elution process is shown in Table 1 below:

[0057] Table 1 Mobile phase elution parameters

[0058]

[0059] 2.2.2 Mass spectrometry conditions

[0060] It was operated in the positive electrospray ionization mode, the spray voltage was 3500 V, the ion transfer tube temperature was 350 °C, the vaporization temperature was 400 °C, the sheath gas flow rate was 50 L / min, the auxiliary gas flow rate was 15 L / min, and the scan gas flow rate was 1 L / min;

[0061] The MRM conditions for eravacycline: the m / z of the precursor ion was 559.2, the m / z of the product ion was 542.2, and the collision energy was 21 V; the MRM conditions for deuterated eravacycline were as follows: the m / z of the precursor ion was 567.21, the m / z of the product ion was 550.3, and the collision energy was 20 V.

[0062] 2.3 Standard curve establishment

[0063] The standard curve includes seven concentration points S1 to S7. The method for establishing the standard curve is as follows: accurately weigh the elacycline standard in a 1 mL brown volumetric flask, dissolve it evenly with methanol and dilute it to 1 mL to prepare an elacycline stock solution with a concentration of 1 mg / mL for standby use; dilute the elacycline stock solution with 50% methanol / water to a standard curve working solution with a mass concentration of 500, 1000, 2500, 5000, 10000, 30000, and 50000 ng / mL,

[0064] Accurately pipette 270 μL of blank serum into a 1.5 mL EP tube, add 30 μL of the standard curve working solution of eraciclib respectively, vortex to mix, and prepare 7 standard curve samples with mass concentrations of 50, 100, 250, 500, 1000, 3000, and 5000 ng / mL. According to the sample pretreatment method, the above seven standard samples were treated and measured, and the standard curve was established with the peak area ratio of the test compound to the internal standard as the Y-axis and the reference substance concentration as the X-axis.

[0065] The ratio of the peak area of elacycline measured in the serum sample to the peak area of the internal standard is brought into the accompanying calibration curve to calculate the content of elacycline in the serum sample.

[0066] 3. Methodological Validation

[0067] The method was validated according to the guidelines for bioanalytical method validation and sample analysis in the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonized Guidelines, including selectivity, carryover effect, linearity, limit of quantitation, accuracy and precision, matrix effect and stability.

[0068] 3.1 Selectivity and carryover effects

[0069] Selectivity is the ability of the analytical method to distinguish and determine the analyte in the presence of potential interfering substances in the blank biological matrix. Double blank serum (without analyte and internal standard), blank serum (with internal standard), lower limit of quantification (LLOQ) and clinical samples were selected, processed and injected, and their chromatograms were analyzed. The response of the interfering peak should not be greater than 20% of the LLOQ peak area, and the peak area at the internal standard retention time should not be greater than 5% of the internal standard peak area.

[0070] A blank sample was injected again after the ULOQ sample to analyze the residual effect. The LLOQ sample, upper limit of quantification (ULOQ) sample and blank sample were injected in sequence to evaluate the residual effect of the method. The peak area observed in the blank serum after the ULOQ sample was less than 20% of the LLOQ peak area and 5% of the internal standard peak area, indicating that the residual of the analyte and internal standard of this method met the requirements (Figure 1 ).

[0071] 3.2 Linear and quantitative lower limit

[0072] Perform LC-MS / MS analysis on the processed serum standard curve samples. Inject samples from low concentration to high concentration and record the chromatogram. The abscissa is the concentration of eravacycline, and the ordinate is the ratio of the peak areas of eravacycline and the internal standard. Take 1 / x 2 as the weighting factor and perform linear regression calculation of the standard curve equation by the least squares method. The lowest detection limit of eravacycline in this method is 50 ng / mL. The standard curve equation of eravacycline is y = 0.0005393x - 0.00556 (r 2 = 0.9959), where y represents the ratio of the peak areas of eravacycline and the internal standard, and x represents the concentration of eravacycline. The results show that eravacycline has a good linear relationship in the range of 50 - 5000 ng / mL, and the correlation coefficient (r 2 ) is greater than 0.99, indicating a good linear relationship ( Figure 2 ).

[0073] 3.3 Precision and accuracy

[0074] Precision refers to the closeness of the concentration values of the target analyte determined repeatedly by this analytical method, which is expressed by the relative standard deviation (RSD%). Accuracy refers to the ratio of the actually measured concentration value to the known theoretical concentration value by this method, which is expressed by the relative error (RE%). Take serum quality control samples at four concentrations of LLOQ, QCL, QCM, and QCH. After pretreatment, perform LC-MS / MS analysis, and determine the accuracy and precision within-day and between-day respectively.

[0075] Continuously measure the low, medium, high concentration and lower limit of quantification quality control samples for 3 days. Use RSD to represent precision and RE to represent accuracy. The within-day accuracy of the quality control samples using this method is between 90.84% and 113.36%, the within-day precision is between 0.27% and 11.01%, the between-day accuracy is between 87.64% and 109.32%, and the between-day precision is between 2.38% and 11.01%. The accuracy and precision of this method are good. See Table 2 below for details:

[0076] Table 2 The lowest quantification limit, accuracy and precision of eravacycline in human serum determined by LC-MS / MS method

[0077]

[0078] 3.4 Matrix effect and recovery

[0079] Take working solutions with two concentrations of QCL and QCH, each take 30 μL, add them to 270 μL of six serum samples from different sources respectively, mix well, and perform sample injection and determination after pretreatment. Each concentration is determined in parallel for 3 replicates. For the matrix of each source, the accuracy (RE) should be within ±15% of the labeled concentration, and the precision (RSD) should not be greater than 15%. The results show (Table 3) that the accuracy of the quality control samples from 6 different sources is between 85.27% - 114.33%, and the precision is between 0.84% - 5.92%, all meeting the requirements.

[0080] Table 3 Matrix effect of ilarcycline in human serum determined by LC-MS / MS

[0081]

[0082] Prepare serum samples with concentrations of 200, 2000, and 4000 ng / mL from the working solutions of QCL, QCM, and QCH. Subsequently, perform the treatment according to the method described in the "2.1 Serum sample pretreatment" section, and determine the peak areas of ilarcycline and the internal standard at each concentration. By substituting the peak area ratio of ilarcycline to the internal standard in the serum sample into the standard curve, calculate the sample concentration, and compare it with the known added concentration to calculate the relative recovery rate.

[0083] Table 4 Relative recovery rate of ilarcycline in human serum determined by LC-MS / MS

[0084]

[0085] 3.5 Stability

[0086] In this experiment, the stability data at room temperature (24 °C) for 24 h, in the injection chamber (4 °C) for 12 h, frozen storage (-20 °C) for 2 d, frozen storage (-80 °C) for 2 d, frozen storage (-20 °C) for 7 d, frozen storage (-80 °C) for 7 d, and the stability after three cycles of freeze-thaw were investigated. The results are shown in Table 4. Take the quality control samples with two concentrations of QCL and QCH and thaw them at room temperature, with 3 replicates for each sample. After pretreatment, perform sample injection and detection. The deviation of the mean value of each concentration from the labeled concentration should be within the range of ±15%. This method has good stability.

[0087] Table 5 Stability of ilarcycline in human serum determined by LC-MS / MS

[0088]

[0089] 3.6 Clinical application of the detection method

[0090] Serum samples of patients in our hospital who used elacycline for anti-infection treatment were collected, and the dosage regimen was 50 mg q12h. After continuous administration to reach a steady-state blood concentration, three serum samples were collected 1 hour before medication to determine the trough concentration. The patient's serum samples were pretreated according to the above-mentioned "serum sample pretreatment" method, detected using the HPLC-MS / MS method, and the accompanying standard curve was established to determine its concentration, and quality control samples were monitored. The measured blood concentrations of elacycline were 330.76 ng / mL, 212.59 ng / mL, and 308.04 ng / mL, respectively. The method provided by this method for determining elacycline in human serum can be used for clinical elacycline concentration detection, which can meet the clinical elacycline blood concentration detection needs.

[0091] The unexplained parts involved in the present invention are the same as the prior art or are implemented by the prior art. The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the raw materials of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the blood drug concentration of eravacycline in serum, characterized in that It includes the following steps: A) Pretreatment of serum samples: The protein components in the serum are removed by the protein precipitation method; B) The content of eravacycline in the serum sample to be detected after pretreatment is detected by liquid chromatography-mass spectrometry. The isotope of eravacycline is used as the internal standard, and the content of eravacycline is calculated by the standard curve method. Among them, the chromatographic conditions are as follows: The chromatographic column is an Acquity UPLC BEH C18 column; the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is ultrapure water containing 0.1% formic acid, and mobile phase B is acetonitrile; the needle washing solution is 50% methanol-aqueous solution; the column temperature is maintained at 45 °C, the injection volume is 2 μL; the mobile phase flow rate is set at 0.3 mL / min, and the total running time is 4.5 minutes. The gradient elution process is as follows: 0 - 0.5 minutes, 1 - 30% B; 0.501 - 1.5 minutes, 30 - 98% B; 1.501 - 2.5 minutes, 98% B; 2.501 - 4.5 minutes, 1% B; The mass spectrometry conditions are as follows: It is operated in the positive electrospray ionization mode, the spray voltage is 3500 V, the ion transfer tube temperature is 350 °C, the vaporization temperature is 400 °C, the sheath gas flow rate is 5.6 L / min, the auxiliary gas flow rate is 10 L / min, and the scan gas flow rate is 1.5 L / min; The MRM conditions for eravacycline: The m / z of the precursor ion is 559.2, the m / z of the product ion is 542.2, and the collision energy is 21 V.

2. The method for detecting the blood drug concentration of eravacycline in serum according to claim 1, characterized in that: Among them, In step A), 60% acetonitrile aqueous solution is selected as the precipitant to remove impurities in the serum; The method for pretreatment of serum samples is as follows: A certain amount of serum is placed in a microcentrifuge tube, an equal volume of internal standard solution is precisely aspirated, and 8 times the volume of the serum of acetonitrile aqueous solution is aspirated. After vortexing for 1 min, it is centrifuged at a high speed of 14000 rpm / min for 5 min by a centrifuge. Subsequently, a certain amount of supernatant is transferred to an injection vial for quantitative analysis by liquid chromatography-mass spectrometry; among them, the volume of the serum is 1 / 150 of the capacity of the microcentrifuge tube.

3. The method for detecting the blood drug concentration of eravacycline in serum according to claim 1, characterized in that: Among them, In step B), the internal standard is selected from deuterated eravacycline, The MRM conditions for deuterated eravacycline are as follows: The m / z of the precursor ion is 567.21, the m / z of the product ion is 550.3, and the collision energy is 20 V.

4. The method for detecting the blood drug concentration of eravacycline in serum according to claim 1, characterized in that: Among them, In step B), the standard curve includes seven concentration points S1 to S7. The method for establishing the standard curve is as follows: accurately weigh the elacycline standard in a 1 mL brown volumetric flask, dissolve it evenly with methanol and dilute it to 1 mL to prepare an elacycline stock solution with a concentration of 1 mg / mL for standby use; dilute the elacycline stock solution with 50% methanol / water to a standard curve working solution with a mass concentration of 500, 1000, 2500, 5000, 10000, 30000, and 50000 ng / mL, Accurately pipette 270 μL of blank serum into a 1.5 mL EP tube, add 30 μL of the standard curve working solution of eraciclib respectively, vortex to mix, and prepare 7 standard curve samples with mass concentrations of 50, 100, 250, 500, 1000, 3000, and 5000 ng / mL. According to the sample pretreatment method, the above seven standard samples were treated and measured, and the standard curve was established with the peak area ratio of the test compound to the internal standard as the Y-axis and the reference substance concentration as the X-axis.

5. The method for detecting the blood concentration of eracycline in serum according to claim 1, characterized in that: Among them, In step B), the ratio of the peak area of elacycline measured in the serum sample to the peak area of the internal standard is brought into the accompanying calibration curve to calculate the content of elacycline in the serum sample.

6. The method for detecting the blood drug concentration of ilarcycline in serum according to claim 1, Features: in, Quality control solution and internal standard working solution are also required during the detection process. The preparation method of the quality control solution is as follows: prepare a 1 mg / mL elacycline stock solution, dilute the elacycline stock solution with 50% methanol / water to QC working solutions with mass concentrations of 2000, 20000, and 40000 mg / mL; The QC working solution was used to prepare the lower limit of quantitation sample LLOQ with a mass concentration of 50 ng / mL and the low quality control samples QCL, medium quality control samples QCM, and high quality control samples QCH with a mass concentration of 200, 2000, and 4000 ng / mL; The concentration of the internal standard working solution is 1500 ng / mL.

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