Method for simultaneously detecting imipenem, relebactam and cilastatin and application thereof

Abstract

The application belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting imipenem, relebactam and cilastatin and application thereof. The method comprises the following steps: adding an internal standard solution to a to-be-detected body fluid sample or a standard curve working solution in the presence of an acetonitrile precipitant, performing solid-liquid separation, taking supernatant, and then detecting the imipenem, relebactam and cilastatin in the to-be-detected body fluid solution by using a liquid chromatography tandem mass spectrometry method; the liquid chromatography tandem mass spectrometry method comprises the following liquid chromatography conditions: using 4-6 mM ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution. The detection method can quickly, simply and accurately detect the concentration levels of imipenem, relebactam and cilastatin in serum, abdominal fluid and urine, and realizes the simultaneous therapeutic drug monitoring of imipenem, relebactam and cilastatin.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and specifically relates to a method for simultaneously detecting imipenem, leribactam and cilastatin and its application. Background Technology

[0002] Imipenem, chemical formula: C 12 H 17 N3O4S, Relebactam (CAS: 1174018-99-5), chemical formula: C 12 H 20 N4O6S, Cilastatin, chemical formula: C 16 H 26 N2O5S. Imipenem is a broad-spectrum carbapenem antibiotic with antibacterial activity against a variety of Gram-positive, Gram-negative, and anaerobic bacteria. Relibactam is a diazabicyclooctane inhibitor with broad-spectrum anti-β-lactamase activity, including class A (such as extended-spectrum β-lactamases and Klebsiella pneumoniae carbapenemases) and class C (AmpC enzymes). It itself has no antibiotic activity but can enhance the activity of β-lactam antibiotics (such as imipenem) and is used to treat infections caused by certain drug-resistant Gram-negative bacteria. Cilastatin is a reversible inhibitor of renal dehydropeptidase-I. It also has no antibiotic activity but exists as a protectant for imipenem, which is rapidly degraded and inactivated in the body by an enzyme in the kidneys (renal dehydropeptidase-I). Cilastatin can inhibit this enzyme, thereby greatly increasing the effective concentration of imipenem in urine and kidneys, allowing it to exert its intended antibacterial effect, and reducing the potential nephrotoxicity of imipenem.

[0003] In clinical practice, imipenem, leribacane, and cilastatin are novel antibiotic combinations used to treat severe infections caused by specific drug-resistant bacteria. This combination is marketed as Ricobra (imipenem for injection). However, its clinical application faces two major challenges: significant pharmacokinetic (PK) variability and the increasingly serious problem of drug resistance. Existing studies have shown that the PK parameters of imipenem in healthy individuals are well-defined, but in special populations (such as critically ill patients receiving ECMO support or those with renal insufficiency), its volume of distribution, clearance, and protein binding rate can change significantly, leading to large fluctuations in blood drug concentrations under conventional dosing regimens, making it difficult to achieve ideal pharmacodynamic targets or causing adverse reactions such as neurotoxicity. Furthermore, there is currently a lack of comprehensive therapeutic drug monitoring protocols for this combination, hindering real-time guidance for clinical medication.

[0004] Therapeutic drug monitoring (TDM), which involves detecting drug concentrations in the blood, is of great significance in clinical treatment. TDM guides clinicians to adjust dosing regimens based on changes in blood drug concentrations and pharmacokinetic parameters. This helps improve drug efficacy while reducing or avoiding toxic reactions. TDM is particularly important for drugs with a narrow therapeutic index and for drugs whose toxic symptoms are easily confused with the disease itself. Through TDM, clinicians can more accurately determine the dosage and frequency of administration, thereby improving the effectiveness and success rate of treatment.

[0005] Imipenem, leribactam, and cilastatin, as a novel antimicrobial combination, present significant pharmacokinetic variability and resistance management challenges in clinical application. Current research on therapeutic drug monitoring (TDM) for this combination is insufficient. Existing techniques mostly involve measuring one or two drugs separately, which is cumbersome, inefficient, and cannot simultaneously detect the concentrations of all three drugs (imipenem, leribactam, and cilastatin), failing to meet the urgent clinical demand for high-throughput and real-time monitoring. Furthermore, for drug penetration data from deep infection sites (such as the peritoneum), reliable analytical methods capable of handling complex matrices (such as abdominal drainage) are needed, as existing analytical methods are significantly affected by these complex matrices.

[0006] Therefore, there is an urgent need in this field to develop an analytical method that can simultaneously detect the concentrations of imipenem, leribactam, and cilastatin in complex matrices (such as serum, peritoneal fluid, and urine) to fill the current gap in TDM research and provide key technical support for precision medicine in clinical practice. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the simultaneous detection of imipenem, leribactam, and cilastatin, and its application, so as to establish an analytical method for the simultaneous, rapid, and accurate determination of the drug concentrations of imipenem, leribactam, and cilastatin in serum, peritoneal fluid, and urine.

[0008] The first aspect of this invention provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin, comprising the following steps: 1) Mix blank body fluid with standard solutions of imipenem, lelibactam, and cilastatin to prepare standard curve working solutions of a series of concentrations; at the same time, prepare two quality control working solutions of different concentrations using the same method; add internal standard solution to the test body fluid sample, standard curve working solution, and quality control working solution respectively in the presence of acetonitrile precipitant, and after solid-liquid separation, take the supernatant to obtain the test body fluid solution, standard curve solution, and quality control solution; 2) The contents of the test body fluid solution, standard curve solution and quality control solution were detected by liquid chromatography-tandem mass spectrometry, and the contents of imipenem, leribactam and cilastatin in the test body fluid solution were calculated. The liquid chromatography conditions for the liquid chromatography-tandem mass spectrometry method include: A CORTECS® HILIC column (2.1 mm × 100 mm, 2.7 μm, Waters) was used. 4-6 mM ammonium acetate aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Different volumes of mobile phase A and mobile phase B were mixed for gradient elution.

[0009] In some optional embodiments, the liquid chromatography conditions described in step 2) further include a column temperature of 35-45 °C and an injection volume of 4-6 μL; And / or, the gradient elution procedure described in step 2) includes: From 0 to 0.5 min, the volume content of mobile phase A was maintained at 10%, the volume content of mobile phase B was maintained at 90%, and the flow rate was 0.4 mL / min; From 0.5 min to 1.5 min, the volume content of mobile phase A was adjusted from 10% to 40%, and the volume content of mobile phase B was adjusted from 90% to 60%, with a flow rate of 0.4 mL / min. From 1.5 min to 3.5 min, the volume content of mobile phase A was maintained at 40%, the volume content of mobile phase B was maintained at 60%, and the flow rate was 0.4 mL / min. From 3.5 min to 3.6 min, the volume content of mobile phase A was adjusted from 40% to 10%, and the volume content of mobile phase B was adjusted from 60% to 90%, with a flow rate of 0.4 mL / min. From 3.6 min to 5 min, the volume content of mobile phase A was maintained at 10%, the volume content of mobile phase B was maintained at 90%, and the flow rate was 0.4 mL / min.

[0010] In some alternative embodiments, the body fluid includes at least one of blood, peritoneal fluid, and urine; The blood includes at least one of serum, plasma, and whole blood; And / or, the body fluid sample to be tested is a body fluid sample taken after taking a drug preparation containing imipenem and / or leribactam and / or cilastatin; And / or, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: The ion spray voltage is 5000-5500 V, the temperature is 450-550 ℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 40-60 psi, the air curtain gas pressure is 30-50 psi, and the collision gas pressure is 5-15 psi.

[0011] In some optional embodiments, the internal standard solution in step 1) is a mixed solution of internal standard and methanol aqueous solution; The volume concentration of methanol in the methanol-water solution is 45-55%; And / or, the volume ratio of the test body fluid sample or standard curve working solution, internal standard solution and acetonitrile precipitant is (90-110):(9-11):(190-210).

[0012] In some alternative embodiments, the internal standard solution contains imipenem-[ 2 H4] and cilastatin-[ 15 N, 2 H3]; And / or, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: The ion spray voltage is 5500 V, the temperature is 500 ℃, the atomizing gas pressure is 50 psi, the auxiliary heating gas pressure is 50 psi, the air curtain gas pressure is 40 psi, and the collision gas pressure is 10 psi.

[0013] Imipenem-[ 2 [H4] is a deuterated imipenem, i.e., a stable isotope-labeled analog formed by replacing four hydrogen atoms in the imipenem molecule with deuterium atoms; cilastatin-[ 15 N, 2 [H3] indicates that in the cilastatin molecule, one nitrogen atom is... 15 N is substituted, and three hydrogen atoms are replaced. 2 H (deuterium) substitution.

[0014] In some alternative implementations, calculating the levels of imipenem, leribactam, and cilastatin in the body fluid sample to be tested includes: The peak areas of imipenem, leribactam, cilastatin, and internal standard in the standard curve working solution were collected; the peak areas of imipenem, leribactam, cilastatin, and internal standard in the body fluid sample to be tested were also collected. Imipenem standard curve was plotted with the concentration of imipenem in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​imipenem in the working solution of the standard curve to the peak area of ​​the internal standard as the ordinate, and the linear regression equation of imipenem was obtained. Then, the ratio of the peak area of ​​imipenem to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation of imipenem to obtain the content of imipenem in the body fluid sample to be tested. The standard curve of lelibazin was plotted with the concentration of lelibazin in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​lelibazin in the working solution of the standard curve to the peak area of ​​the internal standard as the ordinate, and the linear regression equation of lelibazin was obtained. Then, the ratio of the peak area of ​​lelibazin to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation of lelibazin to obtain the content of lelibazin in the body fluid sample to be tested. A standard curve for cilastatin was plotted with the concentration of cilastatin in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​cilastatin in the working solution to the peak area of ​​the internal standard as the ordinate, and the linear regression equation for cilastatin was obtained. Then, the ratio of the peak area of ​​cilastatin to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation for cilastatin to obtain the content of cilastatin in the body fluid sample to be tested.

[0015] In some alternative implementations, the series of concentrations in step 1) includes at least 8 concentrations; And / or, the concentrations of imipenem in the two different quality control working solutions include 400 ng / mL and 8 μg / mL, the concentrations of leribactam include 800 ng / mL and 16 μg / mL, and the concentrations of cilastatin include 400 ng / mL and 20 μg / mL.

[0016] In some optional embodiments, when the blank body fluid is blank blood and the body fluid sample to be tested is the blood sample to be tested, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 40 ng / mL-100 μg / mL. When the blank body fluid is blank peritoneal priming fluid and the body fluid sample to be tested is peritoneal priming fluid sample to be tested, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 40 ng / mL-100 μg / mL. When the blank body fluid is blank urine and the test body fluid sample is the test urine sample, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 80 ng / mL-300 μg / mL. In some optional embodiments, when the blank body fluid is blank blood and the test body fluid sample is the test blood sample, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 40 ng / ml, 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, and 100 ug / ml; When the blank body fluid is a blank peritoneal sudden fluid and the body fluid sample to be tested is a peritoneal sudden fluid sample to be tested, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 40 ng / ml, 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, and 100 ug / ml; When the blank body fluid is blank urine and the test body fluid sample is the test urine sample, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, 100 ug / ml, and 300 ug / ml.

[0017] A second aspect of the present invention also provides an application of the above-described method, the application including its use in detecting the content of imipenem and / or leribactam and / or cilastatin in human body fluids.

[0018] Preferably, the application includes the simultaneous detection of imipenem, leribactam, and cilastatin levels in human body fluids.

[0019] Compared with the prior art, the technical solution of the present invention has at least the following advantages: The present invention provides a method for simultaneous detection of imipenem, leribactam, and cilastatin, comprising the following steps: 1) mixing blank body fluid with standard solutions of imipenem, leribactam, and cilastatin to prepare a series of standard curve working solutions; adding internal standard solutions to the test body fluid sample or standard curve working solution in the presence of acetonitrile precipitant, separating solid and liquid, and taking the supernatant to obtain the test body fluid solution and the standard body fluid solution; 2) detecting the test body fluid solution and the standard body fluid solution using liquid chromatography-tandem mass spectrometry, and calculating the content of imipenem, leribactam, and cilastatin in the test body fluid solution; the liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry include: using 4-6 mM ammonium acetate aqueous solution as mobile phase A, using acetonitrile as mobile phase B, and mixing different volumes of mobile phase A and mobile phase B for gradient elution. This invention optimizes the detection conditions for liquid chromatography by limiting the use of 4-6 mM ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B. This enables protein precipitation pretreatment and utilizes the high separation performance of liquid chromatography and the high selectivity and sensitivity of mass spectrometry to achieve precise quantification of target drugs. It can rapidly, simply, and accurately detect the concentration levels of imipenem, leribacane, and cilastatin in serum, peritoneal fluid, and urine, achieving therapeutic drug monitoring (TDM) of imipenem, leribacane, and cilastatin. This provides crucial evidence for personalized clinical dosing and facilitates personalized precision treatment.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the standard curves for imipenem, leribactam, and cilastatin in the serum matrix of Example 1. (A) is a schematic diagram of the standard curve of imipenem in serum matrix, (B) is a schematic diagram of the standard curve of relibatatan in serum matrix, and (C) is a schematic diagram of the standard curve of cilastatin in serum matrix. Figure 2 This is a schematic diagram of the standard curves of imipenem, riribactam, and cilastatin in the peritoneal drainage matrix of Example 2; (A) is a schematic diagram of the standard curve of imipenem in the abdominal drainage matrix, (B) is a schematic diagram of the standard curve of leribatum in the abdominal drainage matrix, and (C) is a schematic diagram of the standard curve of cilastatin in the abdominal drainage matrix. Figure 3 This is a schematic diagram of the standard curves for imipenem, leribactam, and cilastatin in the urine matrix of Example 3. (A) is a schematic diagram of the standard curve of imipenem in urine matrix, (B) is a schematic diagram of the standard curve of leribacone in urine matrix, and (C) is a schematic diagram of the standard curve of cilastatin in urine matrix. Figure 4 The spectrum of imipenem obtained by the final detection of the analyte in the lowest point standard curve working solution (i.e., the limit of quantification (LoQ) point detection) in the serum matrix of Example 1 is shown. 2 The spectra of H4, levebatan, cilastatin, and cilastatin-[ 15 N, 2 The spectrum of H3]; Where (A) is the spectrum of imipenem in the working solution at the lowest point of the serum matrix standard curve, and (B) is the spectrum of imipenem at the lowest point of the serum matrix standard curve. 2 The spectrum of H4], (C) is the spectrum of leribazine at the lowest point of the serum matrix standard curve, (D) is the spectrum of cilastatin at the lowest point of the serum matrix standard curve, and (E) is the spectrum of cilastatin-[H4] at the lowest point of the serum matrix standard curve. 15 N, 2 The spectrum of H3]; Figure 5 The spectrum of imipenem obtained by finally detecting the analyte in the lowest point standard curve working solution (i.e., the limit of quantification (LoQ) point detection) of the peritoneal effusion matrix in Example 2 is shown. 2 The spectra of H4, levebatan, cilastatin, and cilastatin-[ 15 N, 2 The spectrum of H3]; Where (A) is the spectrum of imipenem at the lowest point of the standard curve of the abdominal drainage matrix, and (B) is the spectrum of imipenem at the lowest point of the standard curve of the abdominal drainage matrix. 2 The spectrum of H4], (C) is the spectrum of leribazine at the lowest point of the standard curve of the peritoneal sac matrix, (D) is the spectrum of cilastatin at the lowest point of the standard curve of the peritoneal sac matrix, and (E) is the spectrum of cilastatin-[H4] at the lowest point of the standard curve of the peritoneal sac matrix. 15 N, 2 The spectrum of H3]; Figure 6 The spectral image of imipenem obtained by finally detecting the analyte in the lowest point standard curve working solution (i.e., the limit of quantification (LoQ) point detection) in the urine matrix in Example 3 is shown. 2 The spectra of H4, levebatan, cilastatin, and cilastatin-[ 15 N, 2 The spectrum of H3]; Where (A) is the spectrum of imipenem at the lowest point of the urine matrix standard curve, and (B) is the spectrum of imipenem at the lowest point of the urine matrix standard curve. 2 The spectrum of [H4], (C) is the spectrum of lelibazin at the lowest point of the urine matrix standard curve, (D) is the spectrum of cilastatin at the lowest point of the urine matrix standard curve, and (E) is the spectrum of cilastatin-[H4] at the lowest point of the urine matrix standard curve. 15 N, 2 The spectrum of H3]; Figure 7 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 1; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 8 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 2; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 9 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 3; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 10 The spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Example 1; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 11 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 4; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 12 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 5; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 13 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 6; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 14 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 7; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 15 The spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Example 4; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 16 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 8; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 17 Spectra of imipenem, leribactam, and cilastatin in the serum sample to be tested in Comparative Example 9; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Figure 18 Spectra of imipenem, leribactam, and cilastatin in the serum samples to be tested in Comparative Example 10; (A) is the spectrum of imipenem in the serum sample to be tested, (B) is the spectrum of leribactam in the serum sample to be tested, and (C) is the spectrum of cilastatin in the serum sample to be tested. Detailed Implementation

[0023] Exemplary embodiments of this disclosure will now be described in more detail. It should be understood that this disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this disclosure and to fully convey its scope to those skilled in the art. Any product identical or similar to this invention, derived by any person based on the teachings of this invention or by combining features of this invention with other prior art, falls within the protection scope of this invention.

[0024] The technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are all conventional methods.

[0025] Clinically, the pathophysiological state of critically ill patients receiving extracorporeal membrane oxygenation (ECMO) support leads to significant changes in their pharmacodynamic (PK) parameters, manifested as abnormal fluctuations in drug clearance and volume of distribution. This variation often results in insufficient or excessive exposure to key antibiotics (such as imipenem), leading to treatment failure or drug toxicity. Therapeutic drug monitoring (TDM) is a crucial technology for addressing these clinical challenges. By measuring drug concentrations in patient serum in real time, TDM can precisely guide dosing, ensuring that key pharmacodynamic (PD) targets are met. This is essential for the effective treatment of infections caused by multidrug-resistant bacteria such as Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and Pseudomonas aeruginosa. Furthermore, critically ill patients often have hypoalbuminemia, which can significantly reduce drug protein binding (studies show it can even drop to 0%), leading to a substantial increase in free drug concentrations. By dynamically monitoring free drug concentrations, TDM can effectively avoid the errors of traditional empirical dosing, achieving an optimal balance between efficacy and safety.

[0026] Furthermore, even when imipenem is used in combination with raribactam, resistance subgroups may still emerge due to inoculum effects or strain differences. TDM technology can maintain effective antimicrobial pressure and reduce the selection risk of resistance mutations through real-time dose optimization. For complex situations such as dynamic changes in renal function and ECMO tubing adsorption, the individualized pharmacokinetic curve data provided by TDM can effectively compensate for the predictive biases of population pharmacokinetic models. Therefore, establishing TDM technology for imipenem / raribactam / cilastatin has become a key means to improve the success rate of treating critically ill infected patients.

[0027] Currently, the gold standard for TDM is considered to be liquid chromatography, typically combined with tandem mass spectrometry (LC-MS / MS). LC-MS / MS is a combined detection method that uses liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometer, ionized, and then the mass spectrometer separates the ion fragments according to their mass-to-charge ratio. The mass spectrum is then obtained by the detector. LC-MS / MS offers high selectivity and sensitivity and is increasingly widely used in clinical applications, capable of detecting many biomarkers that are undetectable by traditional methods.

[0028] Imipenem, leribactam, and cilastatin, as a novel antimicrobial combination, present significant pharmacokinetic variability and resistance management challenges in clinical application. Current research on therapeutic drug monitoring (TDM) for this combination is insufficient. Existing techniques mostly involve measuring one or two drugs separately, which is cumbersome, inefficient, and fails to meet the urgent clinical demands for high-throughput and real-time monitoring. Furthermore, for drug penetration data from deep infection sites (such as the peritoneum), reliable analytical methods capable of handling complex matrices (such as peritoneal drainage) are needed, as existing analytical methods are significantly affected by these complex matrices.

[0029] Therefore, this invention establishes an analytical method based on LC-MS / MS technology that can simultaneously, rapidly, and accurately determine the concentrations of imipenem, leribactam, and cilastatin in human serum, peritoneal effusion, and urine samples.

[0030] The methods for preparing the mobile phase solution, standard solution, internal standard solution, standard curve working solution, and quality control working solution used in all embodiments and comparative examples of this invention include: I. Preparation of mobile phase solution 1. Prepare mobile phase A Mix 5 mL of 1 M ammonium acetate with ultrapure water in a 1000 mL volumetric flask, and dilute to volume with ultrapure water to obtain 1000 mL of 5 mM ammonium acetate aqueous solution (taking the preparation of 1 L mobile phase A as an example, when reducing the volume, the amount of each component added must be reduced proportionally). After ultrasonically shaking for 5 minutes to remove the gas, it can be used. Store at 20℃~25℃, and the shelf life is one week.

[0031] 2. Prepare mobile phase B Take 1000 mL of acetonitrile (taking the preparation of 1 L of mobile phase B as an example, reduce the amount of each component added proportionally when preparing the reduced volume), and use it after ultrasonic vibration for 5 minutes to remove the gas. Store at 20℃~25℃, and the shelf life is one month.

[0032] II. Preparation of standard solutions, internal standard solutions, standard curve working solutions, and quality control working solutions Imipenem, Relebactam, Imipenem-[ 2 H4](Imipenem-[ 2 H4]), Cilastatin, Cilastatin-[ 15 N, 2 H3](Cilastatin-[ 15 N, 2 All H3) were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.

[0033] 1. Preparation of standard solutions and internal standard solutions Imipenem, leribactam, and cilastatin were dissolved in pure water to prepare initial standard solutions for imipenem, leribactam, and cilastatin, respectively. Imipenem-[…] were dissolved in a 50% (v / v) methanol aqueous solution. 2 H4] and cilastatin-[ 15 N, 2 H3], prepare solutions of imipenem and cilastatin separately, and mix them to obtain imipenem-[ 2 H4] and cilastatin-[ 15 N, 2 A mixed solution of H3], i.e., an internal standard solution.

[0034] 2. Prepare standard curve working solutions (BSTD1-BSTD8 / PSTD1-PSTD8) of imipenem, leribactam, and cilastatin at various concentrations in serum matrix / peritoneal fluid matrix, and imipenem, leribactam, and cilastatin at various concentrations in urine matrix, respectively. (1) Prepare serum matrix standard curve working solution (BSTD1-BSTD8) BSTD8: 30 μL imipenem (1 mg / mL) + 30 μL leribactam (1 mg / mL) + 30 μL cilastatin (1 mg / mL) + 210 μL blank serum. The final concentration of imipenem, leribactam, or cilastatin as a single substance is 100 μg / mL.

[0035] BSTD7: 150 μL BSTD8 + 150 μL blank serum, with a final concentration of 50 μg / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0036] BSTD6: 120 μL BSTD7 + 180 μL blank serum, with a final concentration of 20 μg / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0037] BSTD5: 50 μL BSTD6 + 150 μL blank serum, with a final concentration of 5 μg / mL for imipenem, leribactam, or cilastatin.

[0038] BSTD4: 50 μL BSTD5 + 200 μL blank serum, with a final concentration of 1 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0039] BSTD3: 120 μL BSTD4 + 180 μL blank serum, with a final concentration of 400 ng / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0040] BSTD2: 50 μL BSTD3 + 200 μL blank serum, with a final concentration of 80 ng / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0041] BSTD1: 100 μL BSTD2 + 100 μL blank serum, with a final concentration of 40 ng / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0042] (2) Prepare the working solution (PSTD1-PSTD8) for the standard curve of the abdominal effusion matrix. PSTD8: 30 μL imipenem (1 mg / mL) + 30 μL leribactam (1 mg / mL) + 30 μL cilastatin (1 mg / mL) + 210 μL blank peritoneal priming fluid. The final concentration of imipenem, leribactam, or cilastatin as a single substance is 100 μg / mL.

[0043] PSTD7: 150 μL PSTD8 + 150 μL blank peritoneal priming fluid, with a final concentration of 50 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0044] PSTD6: 120 μL PSTD7 + 180 μL blank peritoneal priming fluid, with a final concentration of 20 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0045] PSTD5: 50 μL PSTD6 + 150 μL blank peritoneal priming fluid, with a final concentration of 5 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0046] PSTD4: 50 μL PSTD5 + 200 μL blank peritoneal priming fluid, with a final concentration of 1 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0047] PSTD3: 120 μL PSTD4 + 180 μL blank peritoneal priming fluid, with a final concentration of 400 ng / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0048] PSTD2: 50 μL PSTD3 + 200 μL blank peritoneal priming fluid, with a final concentration of 80 ng / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0049] PSTD1: 100 μL PSTD2 + 100 μL blank peritoneal priming fluid, with a final concentration of 40 ng / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0050] (3) Prepare the working solution (USTD1-USTD8) of the urine matrix standard curve.

[0051] USTD8: 30 μL imipenem (3 mg / mL) + 30 μL leribactam (3 mg / mL) + 30 μL cilastatin (3 mg / mL) + 210 μL blank urine. The final concentration of imipenem, leribactam, or cilastatin as a single substance is 300 μg / mL.

[0052] USTD7: 100 μL USTD8 + 200 μL blank urine, with a final concentration of 100 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0053] USTD6: 150 μL USTD7 + 150 μL blank urine, with a final concentration of 50 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0054] USTD5: 120 μL USTD6 + 180 μL blank urine, with a final concentration of 20 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0055] USTD4: 50 μL USTD5 + 150 μL blank urine, with a final concentration of 5 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0056] USTD3: 50 μL USTD4 + 200 μL blank urine, with a final concentration of 1 μg / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0057] USTD2: 120 μL USTD3 + 180 μL blank urine, with a final concentration of 400 ng / mL for each of the single substances: imipenem, leribactam, or cilastatin.

[0058] USTD1: 50 μL USTD2 + 200 μL blank urine, with a final concentration of 80 ng / mL for each of the following single substances: imipenem, leribactam, or cilastatin.

[0059] The concentrations of imipenem, leribacone, and cilastatin in the working solutions of the serum matrix standard curve and the peritoneal sputum matrix standard curve were 40 ng / ml, 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, and 100 ug / ml, respectively. The concentrations of imipenem, leribacone, and cilastatin in the working solution of the urine matrix standard curve were 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, 100 ug / ml, and 300 ug / ml, respectively.

[0060] 3. Prepare the quality control working solution. (1) Preparation of serum matrix quality control working solution 100 μL of serum matrix standard curve working solution BSTD3 was used as the low-quality control working solution for imipenem in the serum matrix (IMP QCL) and the low-quality control working solution for cilastatin in the serum matrix (CIL-QCL); 240 μL of blank serum was added to 10 μL of serum matrix standard curve working solution BSTD6 to obtain the low-quality control working solution for leribazine in the serum matrix (REP QCL). Take 100 μL of serum matrix standard curve working solution BSTD6 to obtain the serum matrix high-quality control working solution of cilastatin (CIL-QCH); take 115 μL of blank serum and add 10 μL of serum matrix standard curve working solution BSTD8 to obtain the serum matrix high-quality control working solution of imipenem (IMP QCH); take 105 μL of blank serum and add 20 μL of serum matrix standard curve working solution BSTD8 to obtain the serum matrix high-quality control working solution of leribazine (REP QCH).

[0061] The concentrations of imipenem in the above serum matrix quality control working solutions were 400 ng / mL and 8 μg / mL, respectively; the concentrations of leribacone were 800 ng / mL and 16 μg / mL, respectively; and the concentrations of cilastatin were 400 ng / mL and 20 μg / mL, respectively.

[0062] (2) Preparation of peritoneal effusion matrix quality control working solution 100 μL of peritoneal swab matrix standard curve working solution PSTD3 was used as the low-quality control working solution for imipenem in the peritoneal swab matrix (IMP QCL) and the low-quality control working solution for cilastatin in the peritoneal swab matrix (CIL-QCL); 240 μL of blank peritoneal swab was added to 10 μL of peritoneal swab matrix standard curve working solution PSTD6 to obtain the low-quality control working solution for leribacamine in the peritoneal swab matrix (REPQCL). 100 μL of peritoneal swab matrix standard curve working solution PSTD6 was used as the high-quality control working solution of cetirizine in peritoneal swab matrix (CIL-QCH); 115 μL of blank peritoneal swab was added to 10 μL of peritoneal swab matrix standard curve working solution PSTD8 to obtain the high-quality control working solution of imipenem in peritoneal swab matrix (IMP QCH); 105 μL of blank peritoneal swab was added to 20 μL of peritoneal swab matrix standard curve working solution PSTD8 to obtain the high-quality control working solution of leribazine in peritoneal swab matrix (REP QCH).

[0063] The concentrations of imipenem in the above-mentioned peritoneal effusion matrix quality control working solutions were 400 ng / mL and 8 μg / mL, respectively; the concentrations of leribacone were 800 ng / mL and 16 μg / mL, respectively; and the concentrations of cilastatin were 400 ng / mL and 20 μg / mL, respectively.

[0064] (3) Prepare urine matrix quality control working solution Take 100 μL of urine matrix standard curve working solution USTD2 to obtain the urine matrix low-quality control working solution of imipenem (IMP QCL) and urine matrix low-quality control working solution of cilastatin (CIL-QCL); take 240 μL of blank urine and add 10 μL of urine matrix standard curve working solution USTD5 to obtain the urine matrix low-quality control working solution of leribazine (REP QCL). Take 100 μL of urine matrix standard curve working solution USTD5 to obtain the high-quality control working solution of cilastatin in urine matrix (CIL-QCH); take 115 μL of blank urine and add 10 μL of urine matrix standard curve working solution USTD7 to obtain the high-quality control working solution of imipenem in urine matrix (IMP QCH); take 105 μL of blank urine and add 20 μL of urine matrix standard curve working solution USTD7 to obtain the high-quality control working solution of leribazine in urine matrix (REP QCH).

[0065] The concentrations of imipenem in the above-mentioned urine matrix quality control working solutions were 400 ng / mL and 8 μg / mL, respectively; the concentrations of leribacone were 800 ng / mL and 16 μg / mL, respectively; and the concentrations of cilastatin were 400 ng / mL and 20 μg / mL, respectively.

[0066] In this embodiment of the invention, the linearity r of the standard curve should be greater than 0.99, and the accuracy of the low and high quality working fluids should be within ±15% of the theoretical value.

[0067] Example 1 This embodiment provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin, comprising the following steps: 1) Take a 2 mL centrifuge tube and add 100 μL of serum matrix standard curve working solution, 10 μL of internal standard solution, and 200 μL of 100% acetonitrile in sequence. Vortex for 5 min and centrifuge at 12000 rpm for 15 min. Take 100 μL of supernatant as the standard curve solution and transfer it to a 96-well plate for testing.

[0068] Take a 2 mL centrifuge tube and add 100 μL of serum sample to be tested, 10 μL of internal standard solution, and 200 μL of 100% acetonitrile in sequence. Vortex for 5 min and centrifuge at 12000 rpm for 15 min. Take 100 μL of supernatant as the serum solution to be tested and transfer it to a 96-well plate for testing.

[0069] Take a 2 mL centrifuge tube and add 100 μL of serum matrix quality control working solution, 10 μL of internal standard solution, and 200 μL of 100% acetonitrile in sequence. Vortex for 5 min and centrifuge at 12000 rpm for 15 min. Take 100 μL of the supernatant as the quality control solution and transfer it to a 96-well plate for testing.

[0070] 2) The contents of the serum solution to be tested, the standard curve solution and the quality control solution were detected by liquid chromatography-tandem mass spectrometry, and the contents of imipenem, leribactam and cilastatin in the serum solution to be tested were calculated. The conditions for liquid chromatography-tandem mass spectrometry are as follows: 1. Liquid chromatography conditions are shown in Table 1.

[0071] Table 1 Summary of Liquid Chromatography Conditions

[0072] 2. Mass spectrometry conditions are shown in Table 2.

[0073] Table 2 Summary of mass spectrometry conditions

[0074] 3. Ion pair parameters are shown in Table 3.

[0075] Table 3 Summary of ion pair parameters

[0076] For quantitative ion pairs The calculation of the levels of imipenem, leribactam, and cilastatin in the serum solution to be tested includes: The peak areas of imipenem, leribactam, cilastatin, and internal standard in the standard curve working solution were collected; the peak areas of imipenem, leribactam, cilastatin, and internal standard in the serum sample to be tested were also collected. Imipenem concentration in the working solution of the serum matrix standard curve was plotted on the x-axis, and the ratio of imipenem peak area to internal standard peak area in the working solution of the serum matrix standard curve was plotted on the y-axis to obtain the linear regression equation of imipenem. Then, the ratio of imipenem peak area to internal standard peak area in the serum sample to be tested was substituted into the linear regression equation of imipenem to obtain the imipenem content in the serum sample to be tested. The concentration of lelibazin in the working solution of the serum matrix standard curve was plotted on the x-axis, and the ratio of the peak area of ​​lelibazin in the working solution to the peak area of ​​the internal standard was plotted on the y-axis. The linear regression equation of lelibazin was obtained. Then, the ratio of the peak area of ​​lelibazin to the peak area of ​​the internal standard in the serum sample to be tested was substituted into the linear regression equation of lelibazin to obtain the content of lelibazin in the serum sample to be tested. A standard curve for cilastatin was plotted with the concentration of cilastatin in the working solution of the serum matrix standard curve as the abscissa and the ratio of the peak area of ​​cilastatin in the working solution to the peak area of ​​the internal standard as the ordinate, and the linear regression equation for cilastatin was obtained. Then, the ratio of the peak area of ​​cilastatin to the peak area of ​​the internal standard in the serum sample to be tested was substituted into the linear regression equation for cilastatin to obtain the content of cilastatin in the serum sample to be tested.

[0077] This embodiment also provides an instrument operation procedure for a liquid chromatography-mass spectrometry system, including the following steps: Analysis steps: 1. Turn on the computer, enter the Windows operating system, check if the instrument is in good condition, log in to the Analyst software, and enter the workstation main interface.

[0078] 2. Double-click the Hardware configuration button, select LCMS, and then click Activate Profile on the right to activate the instrument. The LCMS module will display a green checkmark, indicating that the connection is successful.

[0079] 3. Double-click "Build Acquisition Batch" in the Acquire tab, select the established method, and edit the sample sequence (sample number, sample tray position, injection volume, etc.) in the sequence editing menu.

[0080] 4. Click Equilibrate in the menu bar, select the previously created liquid chromatography-mass spectrometry method to be run, and set the equilibration time to 10-20 minutes.

[0081] 5. In the Batch Editor, go to the Submit interface on the right, select the sample sequence to be analyzed, and click the Submit button to submit.

[0082] 6. After the equilibration time is over, the instrument status in the lower right corner of the main interface will show "Ready". Click "Start sample" in the menu bar to begin sample analysis and data acquisition.

[0083] 7. Double-click the "Open Data File" option in the "Explore" menu on the left to view the chromatographic images during the detection process at any time.

[0084] 8. After sample collection and analysis, use Mμltiquant to create a quantitative list and perform quantitative analysis.

[0085] Standby steps: 1. Click the Standby button in the menu bar. You will see the flow rate slowly decrease to zero. After the system pressure drops to 0, double-click Hardware configuration. In the pop-up dialog box, select LCMS and then click Deactivateprofile on the right. The LCMS module will display a cross, indicating that the connection can be disconnected.

[0086] 2. Exit the Analyst software and shut down the computer.

[0087] Example 2 This embodiment provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin. The only difference between this method and Example 1 is that in step 1), the serum matrix standard curve working solution is replaced with an equal volume of peritoneal suds matrix standard curve working solution, the serum sample to be tested is replaced with an equal volume of peritoneal suds sample to be tested, and the serum matrix quality control working solution is replaced with an equal volume of peritoneal suds matrix quality control working solution. The other steps are the same as in Example 1.

[0088] Example 3 This embodiment provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin. The only difference between this method and Example 1 is that in step 1), an equal volume of the serum matrix standard curve working solution is replaced with a urine matrix standard curve working solution diluted 10 times with acetonitrile and pure water (volume ratio 2:1), an equal volume of the serum sample to be tested is replaced with a urine sample to be tested diluted 10 times with a mixed solution of acetonitrile and pure water (volume ratio 2:1), and an equal volume of the serum matrix quality control working solution is replaced with a urine matrix quality control working solution diluted 10 times with a mixed solution of acetonitrile and pure water (volume ratio 2:1). The other steps are the same as in Example 1.

[0089] Example 4 This embodiment provides a method for the simultaneous detection of imipenem, leribactam and cilastatin. The only difference between this method and Example 1 is that in step 1), an equal volume of the serum sample to be tested is replaced with another serum sample to be tested. The other steps are the same as in Example 1.

[0090] Comparative Example 1 This comparative example provides a method for the simultaneous detection of imipenem, lelibactam, and cilastatin: the only difference from Example 1 is the use of mobile phase B as acetonitrile:isopropanol (1:1), and the other steps are the same as in Example 1.

[0091] Comparative Example 2 This comparative example provides a method for the simultaneous detection of imipenem, lelibactam, and cilastatin: the only difference from comparative example 1 is the use of a Kinetex F5 column (3 mm × 100 mm, 2.6 μm, Phenomenex) instead of a CORTECS® HILIC column (2.1 mm × 100 mm, 2.7 μm, Waters), the other steps are the same as comparative example 1.

[0092] Comparative Example 3 This comparative example provides a method for the simultaneous detection of imipenem, lelibactam, and cilastatin: the only difference from Comparative Example 1 is the use of a SeQuant® ZIC®-cHILIC column (2.1 mm × 100 mm, 3.0 μm, Merck) instead of a CORTECS® HILIC column (2.1 mm × 100 mm, 2.7 μm, Waters), the remaining steps are the same as Comparative Example 1.

[0093] Comparative Example 4 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Comparative Example 1 is that mobile phase A is a pure aqueous solution, and the remaining steps are the same as those in Comparative Example 1.

[0094] Comparative Example 5 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Comparative Example 1 is that mobile phase A is an aqueous solution containing 0.1 v / v% formic acid, and the remaining steps are the same as those in Comparative Example 1.

[0095] Comparative Example 6 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Comparative Example 1 is that mobile phase A is an aqueous solution containing 10 mM ammonium acetate, and the remaining steps are the same as those in Comparative Example 1.

[0096] Comparative Example 7 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Example 1 is that mobile phase B is methanol, and the other steps are the same as in Example 1.

[0097] Comparative Example 8 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Example 4 is the use of methanol as the precipitant, while the other steps are the same as in Example 4.

[0098] Comparative Example 9 This comparative example provides a method for the simultaneous detection of imipenem, lelibactam, and cilastatin: the only difference from Example 4 is the use of isopropanol as the precipitant, while the other steps are the same as in Example 4.

[0099] Comparative Example 10 This comparative example provides a method for the simultaneous detection of imipenem, leribactam, and cilastatin: the only difference from Example 4 is the use of a precipitant in a 1:1 volume ratio of acetonitrile and isopropanol mixed solution, while the remaining steps are the same as in Example 4.

[0100] Performance verification results of the method 1. Linear Prepared a series of serially diluted standard solutions of known concentrations (100 μg / mL, 50 μg / mL, 20 μg / mL, 5 μg / mL, 1 μg / mL, 400 ng / mL, 80 ng / mL, and 40 ng / mL in serum / peritoneal fluid matrix, and 300 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 5 μg / mL, 1 μg / mL, 400 ng / mL, and 80 ng / mL in urine matrix), added an equal volume of internal standard solution, and used the least squares method to fit the calibration points to form a calibration curve. The fitting result of the serum matrix calibration curve is shown below. Figure 1 As shown, the fitting results of the calibration curve for the abdominal diaphragm matrix are as follows: Figure 2 As shown, the urine matrix calibration curve fitting results are as follows: Figure 3 As shown, the linearity requirement is r>0.99, the deviation of the value from the theoretical value at each concentration point is less than 15%, and the deviation of the detected value from the theoretical value at the limit of quantification (LoQ) point is less than 20%. The experiment obtained that the linear range of imipenem, leribactam, and cilastatin in serum / peritoneal effusion matrix is ​​40 ng / mL~100 μg / mL, LoQ=40 ng / mL, and the linear range of imipenem, leribactam, and cilastatin in urine matrix is ​​80 ng / mL~300 μg / mL, LoQ=80 ng / mL. The standard curves for imipenem in serum matrix were r=0.99893, for reribabane r=0.99846, and for cilastatin r=0.99907; for abdominal fluid matrix, the standard curves for imipenem r=0.99895, for reribabane r=0.99685, and for cilastatin r=0.99936; and for urine matrix, the standard curves for imipenem r=0.99938 and for reribabane r=0. The standard curve for cilastatin was 99861, with r=0.99865. Imipenem, leribactam, and cilastatin showed good linearity in serum, peritoneal effusion, and urine matrices. LoQ point quantitative detection in serum, peritoneal effusion, and urine matrices was accurate. The detected ions were accumulated at the mass spectrometry ion signal acquisition point, resulting in a time-dimensional spectrum. The horizontal axis of the spectrum represents time, and the vertical axis represents the ion signal response intensity (signal intensity). The serum LoQ point quantitative detection spectrum results are shown below. Figure 4 The results of the quantitative detection spectrum of the LoQ point in the abdominal duct fluid are shown below. Figure 5 The results of the quantitative detection spectrum of LoQ points in urine matrix are shown below. Figure 6As shown, this invention exhibits good detection sensitivity and linearity, meeting the needs of clinical testing and scientific research. Mass spectrometry is an important indicator for detecting whether a substance's ions are present, but it is instantaneous; these chromatograms are then used to obtain the final analytical results. 2. Analysis of test results By comparing the detection results of Examples 1 and 4 and Comparative Examples 1-10, it was found that in Comparative Examples 1-3, after the samples underwent the same pretreatment, the relative comparison column was the CORTECS® HILIC column (e.g., ...). Figure 7 As shown), if the chromatographic column is a Kinetex F5 column (3mm × 100mm, 2.6μm, Phenomenex) (as shown) Figure 8 As shown in the figure, cilastatin has a very poor peak shape, and lelibatatan has a low response intensity, making accurate quantification impossible. The comparison column is a CORTECS® HILIC column. If the column were a SeQuant® ZIC®-cHILIC column (2.1mm × 100mm, 3.0μm, Merck) (as shown in the figure), the results would be significantly better. Figure 9 As shown in the figure, cilastatin has a poor peak shape, and the corresponding intensities of imipenem, leribactam, and cilastatin are all low, affecting quantification. Therefore, the CORTECS® HILIC column (2.1mm × 100mm, 2.7μm, Waters) is more effective.

[0101] In Comparative Examples 1 and 4-6, after the samples underwent the same pretreatment, if the mobile phase A was a pure aqueous solution (Comparative Example 4, spectral results are shown in...), Figure 11 The relative mobile phase A was a 5 mM ammonium acetate aqueous solution (Comparative Example 1, spectral results are shown in [reference]). Figure 7 As can be seen, the peak shapes of imipenem and leribactam are extremely poor, and the peak shape of cilastatin is also poor. Furthermore, the corresponding intensities of imipenem, leribactam, and cilastatin are all low, making accurate quantification impossible. If mobile phase A is a 0.1% formic acid aqueous solution (Comparative Example 5, spectral results are shown in...),... Figure 12 The mobile phase A was a 5 mM ammonium acetate aqueous solution (Comparative Example 1, spectral results are shown in [reference]). Figure 7 As can be seen, imipenem has a poor peak shape and extremely low intensity, leiribactam has an extremely poor peak shape and low intensity, and cilastatin has a poor peak shape and low intensity, making accurate quantification impossible; if mobile phase A is a 10 mM ammonium acetate aqueous solution (Comparative Example 6, spectral results see...), Figure 13 Compared with mobile phase A, which is a 5 mM ammonium acetate aqueous solution (Comparative Example 1), it can be seen that lelibactam and cilastatin have poor peak shapes, affecting the quantitative results. Therefore, mobile phase A of 5 mM ammonium acetate aqueous solution is more effective in this invention.

[0102] In Examples 1, 1, and 7, after the samples underwent the same pretreatment, if the mobile phase B was an acetonitrile:isopropanol (1:1) mixed solution (Comparative Example 1, spectrum shown in...), the results showed that... Figure 7 As shown), the relative mobile phase B is acetonitrile (Example 1, spectral results are shown in [reference]). Figure 10 As can be seen, the peak shapes of leribactam and cilastatin are poor, and the corresponding intensities of imipenem, leribactam, and cilastatin are all low, affecting quantification; if mobile phase B is methanol (Comparative Example 7, spectral results are shown in...), Figure 14 Compared to mobile phase B, which uses acetonitrile, lelibactam exhibits extremely poor peak shape, and the corresponding intensities of imipenem, lelibactam, and cilastatin are all low, making accurate quantification impossible. Therefore, acetonitrile is a more effective mobile phase B in this invention.

[0103] In Examples 4 and Comparative Examples 8-10, except for the type of precipitant, the samples underwent the same pretreatment steps. This indicates that when the precipitant is acetonitrile (Example 4, spectral results are shown in [reference needed]), the precipitant is acetonitrile. Figure 15 The precipitant was methanol (Comparative Example 8, spectral results are shown in [reference]). Figure 16 Isopropanol (Comparative Example 9, spectral results are shown below) Figure 17 Acetonitrile:isopropanol (1:1) mixed solution (Comparative Example 10, spectral results are shown in...) Figure 18 As can be seen, imipenem, lelibactam, and cilastatin exhibited the highest corresponding intensities and better peak shapes when acetonitrile was used as the precipitant. Therefore, acetonitrile is the more effective precipitant in this invention.

[0104] 3. Precision and contamination carryover According to Examples 1-3, the prepared serum / abdominal effusion / urine matrix imipenem low and high quality control working solutions were tested three times consecutively for five days. The calculated intraday precision coefficients of variation for serum matrix imipenem were 4.91% and 3.60%, and the interday precision coefficients of variation were 5.85% and 4.03%, respectively; the intraday precision coefficients of variation for abdominal effusion matrix imipenem were 4.55% and 3.05%, and the interday precision coefficients of variation were 3.75% and 2.89%, respectively; and the intraday precision coefficients of variation for urine matrix imipenem were 1.56% and 0.84%, and the interday precision coefficients of variation were 2.97% and 2.50%, respectively. All were less than 15%, demonstrating good precision (see Table 4 for details). The prepared serum / peritoneal sputum / urine matrix-based low and high quality control working solutions of lelibacco were tested three times consecutively over five days. The intraday precision coefficients of variation (CCVs) of lelibacco in the serum matrix were calculated to be 4.97% and 8.20%, and the interday precision CCVs were 3.79% and 5.31%, respectively. The intraday precision CCVs of lelibacco in the peritoneal sputum matrix were 2.36% and 1.73%, and the interday precision CCVs were 8.52% and 9.35%, respectively. The intraday precision CCVs of lelibacco in the urine matrix were 0.56% and 5.77%, and the interday precision CCVs were 2.30% and 7.63%, respectively. All of these values ​​were less than 15%, indicating good precision (see Table 5 for details). The prepared serum / peritoneal sputum / urine matrix cilastatin low and high quality control working solutions were continuously tested three times over five consecutive days. The intra-day precision coefficients of variation (CVA) for cilastatin in the serum matrix were calculated to be 3.39% and 3.16%, and the inter-day CVA were 3.31% and 2.83%, respectively; the intra-day CVA for cilastatin in the peritoneal sputum matrix were 0.26% and 0.29%, and the inter-day CVA were 2.13% and 3.38%, respectively; and the intra-day CVA for cilastatin in the urine matrix were 1.21% and 1.58%, and the inter-day CVA were 5.31% and 2.70%, respectively. All of these values ​​were less than 15%, indicating good precision (see Table 6). Blank samples (50% methanol aqueous solution) were measured immediately after the peak of the calibration curve. The peak area of ​​the blank samples was less than 0.1% of the peak of the calibration curve (see Table 7), demonstrating that the detection method of this invention has no significant carryover contamination.

[0105] Table 4. Precision of imipenem detection

[0106] Table 5. Precision of Detection of Ribavirin

[0107] Table 6. Precision of cilastatin detection

[0108] Table 7. Carryover rates of imipenem, reribactam, and cilastatin

[0109] 4. Accuracy and matrix effect Spike recovery rate: At least five population samples from different sources were collected. Equal volumes of serum, peritoneal fluid, and urine were selected from each sample and mixed. Then, three equal volumes were selected from each of the mixed serum / peritoneal fluid / urine samples. Low / medium / high concentration standard solutions of known concentrations were added to each sample (see Table 8). The same pretreatment and injection analysis procedures as in the previous examples were performed. The analysis was conducted in triplicate. The spike recovery rate % was calculated using the formula: Spike Recovery Rate % = (Measured sample concentration after spike - Measured matrix sample concentration) / Theoretical spike concentration × 100%. The data in Table 8 are shown. The spike recovery rate of imipenem in the serum matrix was 91.50%–99.56%, and the spike recovery rate of leribacane was 9... The spiked recoveries of cilastatin in the peritoneal fluid matrix were 91.81%–103.16%, 97.08%–103.02%, 99.67%–99.78%, and 95.66%–100.25%, respectively. The spiked recoveries of imipenem in the urine matrix were 99.85%–100.85%, 92.10%–93.46%, and 91.58%–99.25%, respectively. These results, as detailed in Table 8, demonstrate the good accuracy of the detection method of this invention.

[0110] Matrix effect: At least five population samples from different sources were collected. Equal volumes of serum, peritoneal lavage fluid, and urine were mixed from each sample. Three equal volumes were then selected from the mixed serum / peritoneal lavage fluid / urine samples, and low, medium, and high concentration standard solutions of known concentrations were added to each (see Table 8). The same pretreatment and injection analysis procedures were performed, and the samples were measured three times in parallel to obtain the average signal intensity (A) at each concentration point. Standard solutions of corresponding low, medium, and high concentration levels were prepared using the same injection reagents, except that the serum / peritoneal lavage fluid / urine samples were replaced with pure aqueous solutions. These solutions were then injected and analyzed to obtain the average signal intensity (B) at each concentration point in the pure solution. Simultaneously, blank serum / bronchoalveolar lavage fluid samples were prepared and measured three times in parallel to obtain the average signal intensity (C) of the blank serum / bronchoalveolar lavage fluid samples. The average signal intensity was calculated based on the peak area ratio of the analyte to the internal standard in each sample group. The matrix effects were calculated using the formula: matrix effect = (peak area ratio of sample A - peak area ratio of sample C) / peak area ratio of sample B * 100%. The matrix effects of imipenem in serum matrix were 87.69%, 92.56%, and 88.76%; for leribacone, 106.86%, 110.77%, and 106.18%; and for cilastatin, 97.63%, 96.20%, and 90.82%. The matrix effects of imipenem in peritoneal fluid matrix were 96.26%, 101.27%, and 88.0%. The matrix effects of leribacone, riribacone, cilastatin, and imipenem in the urine matrix were 96.82%, 93.28%, and 106.91%, respectively; the matrix effects of riribacone, cilastatin, and imipenem in the urine matrix were 102.87%, 105.77%, and 100.54%, respectively; the matrix effects of leribacone, riribacone, and cilastatin in the urine matrix were 92.17%, 90.66%, and 103.26%, respectively; and the matrix effects of cilastatin in the urine matrix were 95.18%, 90.29%, and 104.24%, respectively. These results meet the experimental requirements.

[0111] Table 8 Recovery and matrix effect of imipenem, reribactam and cilastatin

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneous detection of imipenem, leribactam, and cilastatin, characterized in that, Includes the following steps: 1) Mix blank body fluid with standard solutions of imipenem, lelibactam, and cilastatin to prepare standard curve working solutions of a series of concentrations; at the same time, prepare two quality control working solutions of different concentrations using the same method; add internal standard solution to the test body fluid sample, standard curve working solution, and quality control working solution respectively in the presence of acetonitrile precipitant, and after solid-liquid separation, take the supernatant to obtain the test body fluid solution, standard curve solution, and quality control solution; The body fluids include at least one of serum, abdominal fluid, and urine; 2) The contents of the test body fluid solution, standard curve solution and quality control solution were detected by liquid chromatography-tandem mass spectrometry, and the contents of imipenem, leribactam and cilastatin in the test body fluid solution were calculated. The liquid chromatography conditions for the liquid chromatography-tandem mass spectrometry method include: A CORTECS® HILIC column was used, with 4-6 mM ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B. Gradient elution was performed by mixing different volumes of mobile phase A and mobile phase B. The gradient elution process includes: From 0 to 0.5 min, the volume content of mobile phase A is maintained at 10%, and the volume content of mobile phase B is maintained at 90%. From 0.5 min to 1.5 min, the volume content of mobile phase A was adjusted from 10% to 40%, and the volume content of mobile phase B was adjusted from 90% to 60%. From 1.5 min to 3.5 min, the volume content of mobile phase A was maintained at 40%, and the volume content of mobile phase B was maintained at 60%. From 3.5 min to 3.6 min, the volume content of mobile phase A was adjusted from 40% to 10%, and the volume content of mobile phase B was adjusted from 60% to 90%. 3.6 min to 5 min, the volume content of mobile phase A is maintained at 10%, and the volume content of mobile phase B is maintained at 90%.

2. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1, characterized in that, The liquid chromatography conditions described in step 2) also include a column temperature of 35-45 ℃ and an injection volume of 4-6 μL; And / or, the gradient elution procedure described in step 2) includes: 0~0.5 min, elution flow rate is 0.4 mL / min; 0.5 min to 1.5 min, elution flow rate of 0.4 mL / min; Elution time: 1.5 min to 3.5 min, elution flow rate: 0.4 mL / min; 3.5 min to 3.6 min, elution flow rate of 0.4 mL / min; The elution time was 3.6 min to 5 min, with an elution flow rate of 0.4 mL / min.

3. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: The ion spray voltage is 5000-5500 V, the temperature is 450-550 ℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 40-60 psi, the air curtain gas pressure is 30-50 psi, and the collision gas pressure is 5-15 psi.

4. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, The internal standard solution mentioned in step 1) is a mixed solution of internal standard and methanol aqueous solution; The volume concentration of methanol in the methanol-water solution is 45-55%; And / or, the volume ratio of the test body fluid sample or standard curve working solution, internal standard solution and acetonitrile precipitant is (90-110):(9-11):(190-210).

5. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, The internal standard solution contains imipenem-[ 2 H4] and cilastatin-[ 15 N, 2 H3].

6. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, The calculation of the levels of imipenem, leribactam, and cilastatin in the body fluid sample to be tested includes: The peak areas of imipenem, leribactam, cilastatin, and internal standard in the standard curve working solution were collected; the peak areas of imipenem, leribactam, cilastatin, and internal standard in the body fluid sample to be tested were also collected. Imipenem standard curve was plotted with the concentration of imipenem in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​imipenem in the working solution of the standard curve to the peak area of ​​the internal standard as the ordinate, and the linear regression equation of imipenem was obtained. Then, the ratio of the peak area of ​​imipenem to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation of imipenem to obtain the content of imipenem in the body fluid sample to be tested. The standard curve of lelibazin was plotted with the concentration of lelibazin in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​lelibazin in the working solution of the standard curve to the peak area of ​​the internal standard as the ordinate, and the linear regression equation of lelibazin was obtained. Then, the ratio of the peak area of ​​lelibazin to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation of lelibazin to obtain the content of lelibazin in the body fluid sample to be tested. A standard curve for cilastatin was plotted with the concentration of cilastatin in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​cilastatin in the working solution to the peak area of ​​the internal standard as the ordinate, and the linear regression equation for cilastatin was obtained. Then, the ratio of the peak area of ​​cilastatin to the peak area of ​​the internal standard in the body fluid sample to be tested was substituted into the linear regression equation for cilastatin to obtain the content of cilastatin in the body fluid sample to be tested.

7. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, The series of concentrations mentioned in step 1) includes at least 8 concentrations; And / or, the concentrations of imipenem in the two different quality control working solutions include 400 ng / mL and 8 μg / mL, the concentrations of leribactam include 800 ng / mL and 16 μg / mL, and the concentrations of cilastatin include 400 ng / mL and 20 μg / mL.

8. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 1 or 2, characterized in that, When the blank body fluid is blank blood and the body fluid sample to be tested is the blood sample to be tested, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 40 ng / mL-100 μg / mL. When the blank body fluid is blank peritoneal priming fluid and the body fluid sample to be tested is peritoneal priming fluid sample to be tested, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 40 ng / mL-100 μg / mL. When the blank body fluid is blank urine and the test body fluid sample is the test urine sample, the concentration range of imipenem, leribactam or cilastatin in the standard curve working solution is 80 ng / mL-300 μg / mL.

9. The method for simultaneous detection of imipenem, leribactam, and cilastatin according to claim 8, characterized in that, When the blank body fluid is blank blood and the test body fluid sample is the test blood sample, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 40 ng / ml, 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, and 100 ug / ml; When the blank body fluid is a blank peritoneal sudden fluid and the body fluid sample to be tested is a peritoneal sudden fluid sample to be tested, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 40 ng / ml, 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, and 100 ug / ml; When the blank body fluid is blank urine and the test body fluid sample is the test urine sample, the concentration of imipenem, leribactam or cilastatin in the standard curve working solution includes 80 ng / ml, 400 ng / ml, 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, 100 ug / ml, and 300 ug / ml.

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