Method for detecting polymyxin E in plasma based on combination of magnetic bead extraction and liquid chromatography-tandem mass spectrometry

Abstract

The invention provides a method for detecting polymyxin E in plasma based on combination of magnetic bead extraction and liquid chromatography-tandem mass spectrometry, and the method comprises the following steps: by taking polymyxin B1 as an internal standard, pretreating a plasma sample by adopting HLB magnetic beads; and quantifying the content of polymyxin E in the plasma by using liquid chromatography-tandem mass spectrometry. According to the application, the HLB magnetic beads are combined with an automatic treatment system, so that the balancing, extracting and washing steps are simplified, the time and the material cost are remarkably reduced, the pretreatment of the plasma sample is simple and rapid, and interfering substances are removed to the greatest extent; the method shows excellent selectivity and specificity, the average recovery rate is between 99.91 + / -5.45% and 103.1 + / -8.90%, the matrix effect can be ignored, the lowest limit of detection (LLOD) reaches 0.01 mu g / mL, the wide linear range of 0.01-20 mu g / mL is achieved, and a rapid solution is provided for clinical treatment drug monitoring of polymyxin E.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry. Background Art

[0002] Polymyxin E is a cationic peptide antibiotic produced by a Bacillus subspecies called polymyxin. As a cationic peptide, polymyxin E can target the outer membrane of Gram-negative bacteria, destroying the integrity of the lipid membrane, leading to loss of membrane integrity and bacterial death. Polymyxin E is effective against most Gram-negative bacteria and is the last resort for treating multidrug-resistant Gram-negative infections. The main components of polymyxin E are polymyxin E1 and polymyxin E2. Although it has clinical value, due to its narrow therapeutic window, nephrotoxicity and neurotoxicity are the main dose-limiting effects. The target maximum value of the area under the 24-hour steady-state plasma concentration-time curve (AUCss, 24h) of polymyxin E is 50 mg·h·L -1 , equivalent to an average steady-state plasma concentration (Css, avg) of 2 μg / mL. Polymyxins have a narrow therapeutic window, with both efficacy and toxicity closely related to plasma concentration. Therefore, precise therapeutic drug monitoring (TDM) is crucial for reducing the risk of toxicity and preventing antibiotic resistance. Accurate and efficient quantification of polymyxin E in plasma is crucial for guiding personalized treatment plans and improving patient outcomes.

[0003] Traditional methods for quantifying colistin, such as high-performance liquid chromatography (HPLC), immunological methods, and microbiological methods, often suffer from insufficient specificity and low throughput. Furthermore, these techniques may lack the sensitivity required to detect low concentrations of colistin in clinical samples, leading to inaccurate measurements and suboptimal dosing decisions.

[0004] In recent years, liquid chromatography-tandem mass spectrometry (LC-MS) has become a powerful tool for quantifying plasma colistin levels due to its high sensitivity, specificity, and speed. However, the performance of LC-MS is highly dependent on effective sample preparation to remove interfering substances and enrich the target analyte. For the determination of colistin, the ideal sample preparation method should be simple, versatile, and rapid, and maximize the removal of interfering substances. In recent years, LC-MS sample preparation often involves complex sample pretreatment procedures such as protein precipitation, solid phase extraction (SPE), or ultrafiltration combined with precipitation, which can be time-consuming, expensive, and prone to sample loss or contamination.

[0005] Therefore, it is necessary to develop a simple, universal and rapid plasma sample pretreatment method that can remove interfering substances to the greatest extent, as well as a specific, rapid, highly sensitive, accurate and highly stable quantitative analysis method to quantify polymyxin E in plasma, so as to provide the clinic with the required blood concentration data of polymyxin E for monitoring in a timely manner to adjust the drug dosage. Summary of the Invention

[0006] In order to solve the above technical problems, the present application provides a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry.

[0007] The present application provides a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry. The method uses polymyxin B1 as an internal standard, first pre-treating a plasma sample with HLB magnetic beads to obtain a test sample; then using liquid chromatography-tandem mass spectrometry to quantify the content of polymyxin E in the test plasma sample; before pre-treating the plasma sample with the HLB magnetic beads, the HLB magnetic beads are first activated with methanol; The process of activating the HLB magnetic beads with methanol is to disperse the HLB magnetic beads in methanol at a mass volume ratio of 5 mg / ml of HLB magnetic beads to methanol, and vortex mix to obtain an activated magnetic bead solution; In some embodiments, the process of activating the HLB magnetic beads with methanol is as follows: accurately weigh 2 mg of HLB magnetic beads, add 400 μL of methanol, and vortex to mix to obtain a magnetic bead solution at a speed of 2000 rpm for 30 seconds; to obtain an activated magnetic bead solution; The activated high-capacity HLB magnetic beads (2 mg / sample) can improve the adsorption efficiency of low-concentration polymyxin E.

[0008] HLB magnetic beads are magnetic microspheres coated with a hydrophilic-lipophilic balance (HLB) reversed-phase adsorbent. They combine the convenience of magnetic separation with the efficient enrichment capabilities of solid-phase extraction (SPE). They enable automated, high-throughput capture and purification of trace small molecules of medium or low polarity (such as hormones, drugs, and pesticides) from complex biological, environmental, or food samples, allowing for direct LC-MS / MS analysis. This application utilizes HLB magnetic beads composed of a Fe₃O₄ magnetic core coated with a polystyrene matrix and surface-modified with phenyl and pyrrolidone functional groups. The HLB magnetic beads incorporate hydrophilic pyrrolidone functional groups with polar amide bonds (-CON-), which can form strong hydrogen bonds with the carboxyl (-COOH), amino (-NH₂), or peptide bonds (-NH-CO-) of polymyxin E, enhancing hydrophilic adsorption. The hydrophobic polystyrene backbone of the HLB magnetic beads in this application binds to the fatty chains or hydrophobic amino acid side chains of polymyxin E through π-π interactions and van der Waals forces. The phenyl modification of the hydrophobic portion enhances hydrophobicity through π-π stacking with the phenylalanine (Phe) residues or hydrophobic segments of the fatty acid chains of polymyxin E. The HLB magnetic beads used in this application possess both hydrophilic and hydrophobic properties, enabling dual adsorption of polar and non-polar molecules, thereby efficiently and selectively enriching target analytes from complex biological samples.

[0009] This application uses methanol to activate HLB magnetic beads. Methanol can swell HLB polymers, increase the specific surface area, promote the exposure of HLB hydrophobic groups, and promote the binding of hydrophobic fragments of peptides; at the same time, it can optimize the hydrophilicity of the magnetic bead surface and reduce nonspecific adsorption; during the adsorption process of polymyxin E, methanol can reduce sample matrix interference and improve the recovery rate of peptides; at the same time, methanol can activate carboxyl groups and enhance electrostatic / hydrogen bond interactions with peptides.

[0010] The polymyxin E includes polymyxin E1 and polymyxin E2; the content of the polymyxin E is the sum of polymyxin E1 and polymyxin E2; The step of pre-treating the plasma sample with HLB magnetic beads to extract polymyxin E and polymyxin B1 comprises: S1': plasma sample pretreatment: take a plasma sample, add the internal standard working solution and diluent, vortex mix, and obtain the plasma sample pretreatment solution; In some embodiments, the volume ratio of the plasma sample, internal standard working solution and diluent is 5:1:1; The internal standard working solution is 5 μg / mL polymyxin B1 in 0.1% formic acid aqueous solution; the diluent is 2% ammonia aqueous solution; Dilution with 2% ammonia water can reduce the nonspecific binding of polymyxin E to containers / proteins, significantly improving the recovery rate. Synchronously adding polymyxin B1 as an internal standard to the plasma to be tested and extracting polymyxin B1 simultaneously during magnetic bead extraction can compensate for operational variations. Internal standard calibration throughout the process can significantly improve accuracy and precision.

[0011] In some embodiments, the plasma sample is pretreated as follows: 100 μL of plasma to be tested is taken, 20 μL of internal standard working solution and 100 μL of diluent are added, and vortexed to mix; S2': Automated extraction of plasma sample pretreatment solution; the plasma sample pretreatment solution obtained in step S1' is added to a magnetic bead extraction plate preloaded with activated HLB magnetic beads, and the magnetic bead activation, equilibrium, adsorption, washing and elution steps are performed in sequence by a magnetic bead extractor, and the eluate is collected as the sample to be tested; In some embodiments, the automated extraction of the plasma sample pretreatment solution is performed by pre-loading activated magnetic bead solution in the 1st and 7th columns of wells on the magnetic bead extraction plate, pre-loading ultrapure water in the 2nd, 8th, 4th, and 10th columns of wells, pre-loading the plasma sample pretreatment solution prepared in step S1' in the 3rd and 9th columns of wells, pre-loading methanol in the 5th and 11th columns of wells, and pre-loading eluent in the 6th and 12th columns of wells; performing magnetic bead activation, magnetic bead equilibration, sample adsorption, rinsing, cleaning, and elution steps in sequence through a magnetic bead extractor, and finally collecting the eluent in the 6th and 12th columns of wells, and centrifuging the supernatant to obtain the sample to be tested.

[0012] The specific process is as follows: S21' preparation of a magnetic bead extraction plate; the magnetic bead extraction plate is a 96-well deep-well plate with 12 columns and 8 rows; an appropriate amount of activated magnetic bead solution is placed in the wells in the 1st and 7th columns; an appropriate amount of ultrapure water is added to the wells in the 2nd and 8th, 4th and 10th columns; an appropriate amount of methanol is added to the wells in the 5th and 11th columns; an appropriate amount of eluent is added to the wells in the 6th and 12th columns; and the plasma sample pretreatment solution prepared in step S1' is added to the wells in the 3rd and 9th columns; The eluent is a 5% formic acid-30% acetonitrile aqueous solution; Using a 5% formic acid-30% acetonitrile aqueous solution as the eluent can improve elution efficiency. The 5% formic acid reduces the affinity of the target for the HLB magnetic beads through protonation, promoting desorption. Furthermore, the 5% formic acid adjusts the pH to an acidic state, inhibiting the ionization of the target and reducing nonspecific binding to residual silanol groups on the bead surface. The 30% acetonitrile disrupts hydrophobic interactions for efficient elution. Acetonitrile is highly miscible with water, preventing phase separation and ensuring uniform elution. The 5% formic acid-30% acetonitrile aqueous solution is compatible with subsequent LC-MS / MS analysis, as formic acid enhances signal response in positive ion mode. A 30% acetonitrile content reduces solvent effects and improves peak shape. Using a 5% formic acid-30% acetonitrile aqueous solution can reduce matrix interference. Formic acid precipitates residual proteins, while acetonitrile reduces the solubility of phospholipids, synergistically reducing mass spectrometry ion suppression. 5% formic acid-30% acetonitrile aqueous solution as the eluent can balance the adsorption and elution of HLB magnetic beads. 5% formic acid maintains hydrophilic interactions, while 30% acetonitrile regulates hydrophobic elution.

[0013] In some embodiments, a magnetic bead extraction plate is prepared by placing 200 μL of activated magnetic bead solution in the 1st and 7th columns of wells; adding 400 μL of ultrapure water to the 2nd and 8th, 4th and 10th columns of wells; adding 200 μL of methanol to the 5th and 11th columns of wells; adding 200 μL of elution solution to the 6th and 12th columns of wells; and adding an appropriate amount of plasma sample pretreatment solution to the 3rd and 9th columns of wells; S22': Automated extraction: Place the prepared magnetic bead extraction plate in the magnetic bead extractor and perform automated extraction according to the following process: Activation of magnetic beads: shake the magnetic beads in the first and seventh columns for 30 seconds, magnetically absorb for 30 seconds, and transfer to the second and eighth columns; Magnetic bead balance: shake the magnetic beads in the 2nd and 8th columns for 60 seconds, magnetically absorb for 30 seconds, and transfer them to the 3rd and 9th columns; Sample loading: oscillate the magnetic beads in the 3rd and 9th columns for 150 seconds, magnetically absorb for 30 seconds, and transfer to the 4th and 10th columns; Magnetic bead washing: shake the magnetic beads in the 4th and 10th columns for 60 seconds, magnetically absorb for 30 seconds, and transfer them to the 5th and 11th columns; Magnetic bead washing: shake the magnetic beads in the 5th and 11th columns for 60 seconds, magnetically absorb for 30 seconds, and transfer them to the 6th and 12th columns; Magnetic bead elution: The magnetic beads in the 6th and 12th columns are shaken for 150 seconds, magnetically attracted for 30 seconds, and then transferred to the 1st and 7th columns and discarded; the eluate contains the target compound; S23′: centrifuge the eluates in the 6th column well and the 12th column well, and take the supernatant of the eluate as the sample to be tested; During the preparation and automated extraction process of the magnetic bead extraction plate of the present application, activated magnetic bead liquid is placed in the first column of wells and the seventh column of wells to ensure that the binding ability of the magnetic beads to the analyte is consistent; ultrapure water is placed in the second column of wells and the eighth column of wells to balance with the aqueous phase and remove methanol residues; ultrapure water is placed in the fourth column of wells and the tenth column of wells as a weak washing step to remove salts and water-soluble nonspecific adsorption impurities; methanol is placed in the fifth column of wells and the eleventh column of wells as a strong washing step to remove phospholipids and hydrophobic interferences; acidic eluent is placed in the sixth column of wells and the twelfth column of wells to selectively release polymyxin E and polymyxin B1, reduce co-eluted impurities, and ultimately elute the target analyte polymyxin E; during the automated extraction process, the gradient washing design of weak washing (water), strong washing (methanol), and acidic elution is synergistically enhanced with methanol-activated carboxyl-modified HLB magnetic bead extraction to enhance the specificity and selectivity of magnetic bead extraction of polymyxin E in plasma, with good recovery rate and reduced matrix effect.

[0014] The liquid chromatography conditions are as follows: a Kinetex XB-C18 column (length 100 mm × inner diameter 2.1 mm × filler particle size 2.6 μm) or equivalent performance; a column temperature of 40°C; an injection volume of 2 μL; mobile phase A consisting of a 0.2% formic acid-water solution; mobile phase B consisting of a 0.2% formic acid-acetonitrile solution; gradient elution of the mobile phase through the column; a flow rate of 0.35 ml / min. The conditions of the gradient elution are as follows: elution time is 0 minute to 0.5 minute, the volume ratio of mobile phase A: mobile phase B is 96:4; elution time is 0.5 minute to 2 minutes, the volume ratio of mobile phase A: mobile phase B changes from 96:04 to 82:18; elution time is 2 minutes to 3.5 minutes, the volume ratio of mobile phase A: mobile phase B changes from 82:18 to 77:23; elution time is 3.5 minutes to 3.6 minutes, the volume ratio of mobile phase A: mobile phase B changes from 77:23 to 10:90; elution time is 3.6 minutes to 4.6 minutes, the volume ratio of mobile phase A: mobile phase B is maintained at 10:90; elution time is 4.6 minutes to 4.7 minutes, the volume ratio of mobile phase A: mobile phase B changes from 10:90 to 96:4; elution time is 4.7 minutes to 5.5 minutes, and the volume ratio of mobile phase A: mobile phase B is maintained at 96:4; Under the liquid chromatography conditions, polymyxins E1 and E2 were separated from the internal standard polymyxin B1 by retention time, achieving effective chromatographic peak separation; post-column cleaning was performed for 1.2 minutes to thoroughly elute, remove residues, and reduce carryover effects.

[0015] The mass spectrometer uses electrospray ionization positive ion mode to detect the precursor ions and product ions of polymyxin E1, polymyxin E2 and polymyxin B1 to perform quantitative analysis of polymyxin E1 and polymyxin E2; In some embodiments, the mass spectrometry parameters are as follows: ion source temperature of 550° C., nebulizer gas and auxiliary heater gas of 60 psi, curtain gas of 40 psi, collision gas of 9 psi, ion spray voltage of 4000 V; ionization mode is electrospray ionization positive ion mode; quantitative analysis of polymyxin E1 and polymyxin E2 is performed by detecting the precursor ions and product ions of polymyxin E1, polymyxin E2, and polymyxin B1 in electrospray ionization positive ion mode; The quantitative ion pair of polymyxin E1 is 390.900 and 385.000; the qualitative ion pair of polymyxin E1 is 390.900 and 101.100; the quantitative ion pair of polymyxin E2 is 386.100 and 380.100; the qualitative ion pair of polymyxin E2 is 386.100 and 101.100; the quantitative ion pair of polymyxin B1 is 602.569 and 241.1; wherein, the polymyxin The precursor ion of E1 is an ion at 390.900; the product ions of polymyxin E1 are ions at 385.000 and 101.100; the precursor ion of polymyxin E2 is an ion at 386.100; the product ions of polymyxin E2 are ions at 380.100 and 101.100; the precursor ion of polymyxin B1 is an ion at 602.569; the product ion of polymyxin E2 is an ion at 241.1; This application selected specific ion pairs of polymyxin E1 (Q1: 390.9 → Q3: 385.0 / 101.1) and E2 (Q1: 386.1 → Q3: 380.1 / 101.1) to avoid endogenous interference in plasma and achieve specific detection of polymyxin E1 and E2; m / z 385.00 and 380.1 are highly abundant quantitative ions with little interference, which can improve detection sensitivity; m / z 101.1 assists in confirming the identity of the target; the internal standard (polymyxin B1, Q1: 602.569 → Q3: 241.1) corrects for matrix effects and reduces matrix effects; doubly charged ion monitoring enhances signal linearity.

[0016] The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry comprises the following steps: S1: Prepare a series of concentrations of standard working solutions, quality control solutions, and internal standard working solutions; The standard working solution of the series of concentrations is a mixed standard working solution of polymyxin E1 and polymyxin E2 prepared by blank plasma, with a concentration range of 0.01-20 μg / mL; The series of quality control solutions are prepared by using blank plasma to prepare low, medium and high concentrations of polymyxin E1 and polymyxin E2 mixed quality control solutions, with a concentration range of 0.2-8 μg / mL; In some embodiments, the concentrations of the polymyxin E1 and polymyxin E2 mixed standard working solutions include 20.00, 10.00, 5.00, 2.00, 0.50, 0.10, 0.05, and 0.01 μg / mL; the concentrations of the polymyxin E1 and polymyxin E2 mixed quality control solutions include: 0.2, 1, and 8 μg / mL; The internal standard working solution is a polymyxin B1 solution with a concentration of 5 μg / mL prepared with 0.1% formic acid water; This application uses blank plasma to prepare standards, which can offset matrix differences in extraction and ionization efficiency; it has a wider linear range and good linear relationship.

[0017] S2: Pretreatment of standard working solution, quality control solution: Take the standard working solution or quality control solution prepared in step S1, add the internal standard working solution and the diluent, and vortex mix to obtain the pretreatment standard working solution or pretreatment quality control solution; the volume ratio of the standard working solution or quality control solution, the internal standard working solution and the diluent is 5:1:1; In some embodiments, the pretreatment of the standard working solution or the quality control solution is as follows: 100 μL of the standard solution or the quality control solution is taken, 20 μL of the internal standard working solution and 100 μL of the diluent are added, and vortexed to mix, thereby obtaining the pretreated standard working solution or the pretreated quality control solution; S3: Automated extraction of the pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution prepared in step S2 to prepare the standard working solution to be tested, the quality control solution to be tested, and the sample to be tested; the automated extraction of the pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution prepared in step S2 to prepare the standard working solution to be tested, the quality control solution to be tested, and the sample to be tested is based on the automated extraction of the plasma sample pretreatment solution in step S2', and the 3rd and 9th column wells are pre-loaded with the pretreatment standard working solution, pretreatment quality control solution, or the plasma sample pretreatment solution prepared in step S1'; S4: injecting the working solution of the standard sample to be tested prepared in step S3 into liquid chromatography-tandem mass spectrometry for analysis, performing linear regression analysis with the concentration ratio of the standard sample to the internal standard as the X-axis and the peak area ratio as the Y-axis to obtain a standard curve and a standard curve equation;

[0018] S5: injecting the test sample prepared in step S3 into liquid chromatography-tandem mass spectrometry for analysis, and quantifying polymyxin E1 and polymyxin E2 in the test plasma sample based on the standard curve and standard curve equation obtained in step S4;

[0019] S6: Inject the quality control solution to be tested in step S3 into liquid chromatography-tandem mass spectrometry for analysis. Calculate the amount of polymyxin E1 and polymyxin E2 in the quality control solution to be tested based on the standard curve and standard curve equation obtained in step S4, and calculate the accuracy and precision based on the theoretical content of polymyxin E1 and polymyxin E2 in the quality control solution to be tested. When the accuracy and precision deviations are within the predetermined acceptance criteria, the test result of step S5 is valid; if the accuracy and precision deviations exceed the predetermined acceptance criteria, retest according to steps S1 to S6.

[0020] The predetermined acceptance criteria are a deviation within ±15% and an RSD less than 15%; This application ensures the accuracy and reliability of polymyxin E monitoring data through internal standard calibration and full monitoring of quality control products.

[0021] In summary, this application has the following beneficial technical effects: 1. This application presents, for the first time, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS) method based on HLB magnetic bead extraction for the quantification of polymyxin E in plasma. By combining HLB magnetic beads with an automated processing system, equilibration, extraction, and washing are simplified, significantly reducing time and material costs. This method makes plasma sample pretreatment simple and rapid, and maximizes the removal of interfering substances. Combined with optimized LC-MS parameters, this method demonstrates excellent selectivity and specificity in liquid chromatography-tandem mass spectrometry (LC-MS) analysis, with average recoveries ranging from 99.91±5.45% to 103.1±8.90% and negligible matrix effects. The lowest limit of detection (LLOD) reaches 0.01 μg / mL, with a wide linear range of 0.01-20 μg / mL, providing a rapid solution for therapeutic drug monitoring of polymyxin E in clinical practice.

[0022] 2. The technical features of the technical solution of the present application have a synergistic effect. The HLB magnetic beads, MRM specific ion pairs, and liquid chromatography gradient elution technical features adopted in the present application realize the specificity of the method of the present application for the detection of polymyxin E; the present application uses blank plasma to prepare the standard working solution to offset the matrix differences in magnetic bead extraction and ionization efficiency, and combines doubly charged ion monitoring to achieve a wide linear range and a good linear relationship; the present application uses HLB magnetic beads for extraction, 2% ammonia water as a diluent, and high-abundance fragment ions to achieve high sensitivity of detection; the present application adopts automated extraction, internal standard correction, and full-process monitoring of quality control products, so that the present method has good precision and accuracy; the present application uses methanol to activate HLB magnetic beads, and gradient elution is used during the extraction process, and the ion suppression / enhancement effect of polymyxin B1 is simultaneously corrected, so that the method has a good recovery rate and a low matrix effect; in chromatographic separation, post-column cleaning is used to remove residues; there is little carryover effect and good sample detection stability. Compared with commercial SPE columns (Oasis® HLB, 35 yuan / sample), this method reduces the cost per sample by 71% (10 yuan / sample) and improves throughput, making it more suitable for large-scale applications in clinical laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present application will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will appreciate that these drawings are drawn only for the purpose of explaining the preferred embodiments and therefore should not be construed as limiting the scope of the present disclosure. In addition, unless otherwise specified, the drawings are merely schematic representations of the composition or structure of the described objects and may contain exaggerated representations. The drawings are not necessarily drawn to scale.

[0024] Figure 1 Schematic diagram of the structures of polymyxin E1, polymyxin E2, and polymyxin B1; wherein, Figure 1 The formula I portion is a schematic diagram of the structure of polymyxin E1; Figure 1 The formula II portion is a schematic structural diagram of polymyxin E2; Figure 1 The formula III portion is a schematic structural diagram of polymyxin B1.

[0025] Figure 2 Magnetic bead extraction workflow diagram, where A is a schematic diagram of the 96-well plate layout; B is a schematic diagram of the magnetic bead extraction and purification device; and C is a schematic diagram of the magnetic bead extraction process.

[0026] Figure 3 MRM chromatogram of polymyxin E1 in blank plasma sample without polymyxin B1 added.

[0027] Figure 4 MRM chromatogram of polymyxin E2 in blank plasma sample without polymyxin B1.

[0028] Figure 5 MRM chromatogram of polymyxin B1 in blank plasma sample without polymyxin B1 added.

[0029] Figure 6 MRM chromatograms of polymyxin E1 in plasma samples spiked with 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantification).

[0030] Figure 7 MRM chromatograms of polymyxin E2 in plasma samples spiked with 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantification).

[0031] Figure 8 MRM chromatograms of polymyxin B1 in plasma samples spiked with 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantification).

[0032] Figure 9 Chromatogram of polymyxin E1 for carryover effect detection.

[0033] Figure 10 Chromatogram of polymyxin E2 for carriage effect detection.

[0034] Figure 11 Chromatogram of polymyxin B1 for carryover effect detection.

[0035] Figure 12 Chromatogram of polymyxin E1 in patient plasma sample.

[0036] Figure 13 Chromatogram of polymyxin E2 in patient plasma sample.

[0037] Figure 14 Chromatogram of polymyxin B1 in patient plasma sample.

[0038] Figure 15 Chromatogram of polymyxin E1 after protein precipitation pretreatment.

[0039] Figure 16 This is the chromatogram of polymyxin E1 after magnetic bead pre-treatment in this application. DETAILED DESCRIPTION

[0040] The following is combined with Figures 1 to 16 , a detailed description of the present disclosure is given.

[0041] In order to make the purpose, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present disclosure and are not intended to limit the present disclosure.

[0042] Chemicals and reagents Polymyxin E1 sulfate standard, CAS number 1066-17-7, was purchased from Zhengda Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China, batch number 20211118-1, with a purity of 78.4%. The structural formula is as follows Figure 1 As shown in formula I.

[0043] Polymyxin E2 sulfate standard, CAS No. 30387-41-8, was purchased from Zhengda Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China, batch No. 20221203, with a purity of 79.9%. The structural formula is as follows Figure 1 As shown in formula II.

[0044] Internal standard compound (IS): Polymyxin B1 standard, CAS No. 4135-11-9, purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China, product No. P343277-1mg, batch No. F2418155, structural formula Figure 1 As shown in formula III.

[0045] Depend on Figure 1 It can be seen that the fatty acid side chain of polymyxin E1 is 6-methyloctanoic acid, the fatty acid side chain of polymyxin E2 is 6-methylheptanoic acid, the 6th amino acid of polymyxin B1 is phenylalanine, and the 6th amino acid of polymyxin E is leucine.

[0046] Magnetic beads: HLB magnetic beads (30 μm), composed of a Fe3O4 core coated with a polystyrene matrix, surface modified with phenyl and pyrrolidone functional groups, with a specific surface area of ​​680 m² / g and an average pore size of 80 Å, were purchased from Junrong Biotechnology Co., Ltd., Hangzhou, China.

[0047] HPLC grade methanol, acetonitrile, and formic acid were purchased from Fisher Chemical, Fair Lawn, NJ, USA.

[0048] Ultrapure water was prepared using a Milli-Q water purification system from Merck Millipore, Darmstadt, Germany.

[0049] Unless otherwise specified, other reagents used in the examples of this application are from conventional commercial products.

[0050] Instruments and Equipment LC-MS: AB SCIEX Triple Quad™ 4500 mass spectrometer coupled with Jasper™ high performance liquid chromatography system. Data acquisition and statistical analysis were performed using Analyst 1.6.3 software (SCIEX, USA).

[0051] Magnetic bead extractor: Smart 32, purchased from Guangzhou Daan Biotechnology Co., Ltd., China.

[0052] Solution preparation Accurately weigh the polymyxin E1 and polymyxin E2 sulfate standards and prepare them into stock solutions using a 0.1% formic acid-10% methanol solution. Then, dilute the stock solutions with 0.1% formic acid solution to prepare mixed standard stock solutions with concentrations ranging from 10 to 1000 μg / mL. A series of dilutions using blank plasma are then performed to prepare standard working solutions with concentrations ranging from 0.01 μg / mL to 20 μg / mL, as well as low, medium, and high concentration quality control solutions. The specific preparation procedures for the standard working solutions and quality control solutions are as follows: Preparation of standard stock solution Accurately weigh 4 mg of polymyxin E1 sulfate standard and dissolve it in 1568 μL (2 ml * 0.784, the coefficient of the polymyxin E1 sulfate standard purity converted to sulfate mass is 0.784) of 0.1% formic acid-10% methanol aqueous solution to obtain a 2 mg / ml polymyxin E1 standard stock solution.

[0053] Accurately weigh 4 mg of polymyxin E2 sulfate standard and add 1598 μL (2 ml * 0.799, the coefficient of the polymyxin E2 sulfate standard converted to sulfate mass is 0.799) of 0.1% formic acid-10% methanol aqueous solution to dissolve it to obtain a 2 mg / ml polymyxin E2 standard stock solution.

[0054] Accurately weigh 2 mg of polymyxin B1 standard and dissolve it in 1900 μL (2 ml * 0.95, the purity conversion coefficient is 0.95) of 0.1% formic acid-10% methanol aqueous solution to obtain a 1 mg / ml polymyxin B1 standard stock solution.

[0055] Preparation of mixed standard stock solution Take the polymyxin E1 standard stock solution and the polymyxin E2 standard stock solution and prepare them according to Table 1.

[0056] Table 1 Configuration of mixed standard stock solution

[0057] Preparation of standard working solutions and quality control products Working solutions of standards and quality controls were prepared in the same plasma matrix as the test samples. Final standard concentrations of polymyxin E1 and E2 were obtained by appropriate dilution with blank plasma: 20.00, 10.00, 5.00, 2.00, 0.50, 0.10, 0.05, and 0.01 μg / mL. Quality control concentrations of polymyxin E1 and E2 were 0.2, 1, and 8 μg / mL, respectively. Working solutions of standards and quality control samples were prepared according to Table 2.

[0058] Table 2 Preparation of standard working solutions and quality control samples

[0059] Preparation of internal standard working solution Take the polymyxin B1 standard stock solution and then prepare the internal standard working solution according to Table 3.

[0060] Table 3 Preparation of internal standard working solution

[0061] The final concentrations of all solutions mentioned above are expressed as μg / mL of free base. Standard stock solutions, mixed standard stock solutions, standard curve working solutions, and quality control samples were all stored at -80°C.

[0062] Preparation of dilution solution (2% ammonia water) Take 49 ml of pure water and place it in a 50 ml centrifuge tube. Add 1 ml of ammonia water and vortex to mix to obtain the diluent (2% ammonia water).

[0063] Preparation of eluent (5% formic acid-30% acetonitrile water) Take 2.5 ml of formic acid, 15 ml of acetonitrile and 35 ml of pure water and place them in a 50 ml centrifuge tube. Vortex to mix to obtain the eluent (5% formic acid-30% acetonitrile water).

[0064] Preparation of activated magnetic bead solution Accurately weigh 2 mg of HLB magnetic beads, add 400 μL of methanol, and vortex to mix to obtain activated magnetic bead solution. The rotation speed is 2000 rpm and the vortex time is 30 s.

[0065] HLB magnetic beads were used to pre-treat standard working solution, quality control solution, and plasma samples. The pretreatment of the standard working solution, quality control solution, and plasma sample using HLB magnetic beads includes first pretreating the standard working solution, quality control solution, and plasma sample to prepare pretreated standard working solution, pretreated quality control solution, and plasma sample pretreatment solution; then using the HLB magnetic beads to perform automated extraction using a magnetic bead analyzer to obtain the standard working solution to be tested, the quality control solution to be tested, and the sample to be tested. The specific pretreatment process is as follows: Preparation of pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution Take 100 μL of standard working solution, add 20 μL of internal standard working solution and 100 μL of diluent, and vortex to mix to obtain the pretreatment standard working solution.

[0066] Take 100 μL of quality control solution, add 20 μL of internal standard working solution and 100 μL of diluent, and vortex to mix to obtain the pretreated quality control solution.

[0067] Take 100 μL of plasma to be tested, add 20 μL of internal standard working solution and 100 μL of diluent, and vortex to mix to obtain the plasma sample pretreatment solution.

[0068] Preparation of standard working solution, quality control solution and sample to be tested The standard working solution, quality control sample, and sample to be tested are prepared by using HLB magnetic beads and automatically extracted by a magnetic bead instrument. Activated magnetic bead solution is pre-loaded in the 1st and 7th column wells, ultrapure water is pre-loaded in the 2nd, 8th, 4th, and 10th column wells, the 3rd and 9th column wells are pre-loaded with the standard working solution to be tested or the quality control solution to be tested or the plasma sample pretreatment solution, methanol is pre-loaded in the 5th and 11th column wells, and the 6th and 12th column wells are pre-loaded with the eluent on a magnetic bead extraction plate. The magnetic bead activation, magnetic bead equilibration, sample adsorption, elution, cleaning, and elution steps are sequentially performed by the magnetic bead extractor. Finally, the eluent in the 6th and 12th column wells is collected, and the supernatant is centrifuged to obtain the standard working solution to be tested or the quality control solution to be tested or the sample to be tested.

[0069] The process of automated extraction using HLB magnetic beads and a magnetic bead analyzer is as follows: The precipitate was extracted using a magnetic bead extractor (Smart 32, Daan Biotechnology Co., Ltd., Guangzhou, China). Figure 2 The device is equipped with two sets of eight parallel strips, which can accommodate two 96-well deep-well plates (as shown in Figure B). Figure 2 As shown in Figure A, each parallel strip consists of a non-magnetic cap and a magnetic rod. The magnetic rods are extended and retracted to achieve agitation and adsorption of the magnetic beads. The device allows programmable control of the strip's movement between well columns.

[0070] After vortex mixing (2000 rpm, 30 seconds) of the activated magnetic bead solution (2.0 mg of magnetic beads dispersed in 400 μL of methanol), 200 μL of the magnetic bead solution was respectively dispensed into the 1st and 7th columns of wells of a 96-well plate; 400 μL of ultrapure water was added to the 2nd and 8th columns of wells respectively; 200 μL of methanol was added to the 5th and 11th columns of wells respectively; 200 μL of eluent (5% formic acid-30% acetonitrile) was added to the 6th and 12th columns of wells respectively; 100 μL of pretreatment standard working solution or pretreatment quality control product or plasma sample pretreatment solution was taken, 20 μL of internal standard working solution and 100 μL of diluent (2% ammonia water) were added, and after vortex mixing, the mixture was transferred to the 3rd and 9th columns of wells respectively.

[0071] The fully automated analyte extraction process is as follows (e.g. Figure 2 C in Figure 4): The cap was lowered to wells 1 and 7 and vertically shaken for 60 seconds to fully activate and evenly disperse the HLB magnetic beads. A magnetic rod was then inserted into the cap for magnetization, allowing the beads to adsorb for 30 seconds before being transferred to wells 2 and 8 for aqueous equilibration (60 seconds of oscillation and 30 seconds of adsorption). The beads were then transferred to wells 3 and 9 to extract the target polymyxins from the sample (150 seconds of oscillation and 30 seconds of adsorption). The beads were then transferred to wells 4 and 10 for a weak wash (aqueous phase, 60 seconds of oscillation and 30 seconds of adsorption), followed by a strong wash (methanol phase, 60 seconds of oscillation and 30 seconds of adsorption). Finally, the beads were eluted in wells 6 and 12 by shaking for 150 seconds to obtain the eluate. The beads were collected with the magnetic rod for 30 seconds and then discarded in wells 1 and 7 to complete the process. The eluate contains the target substance to be tested. The eluate is centrifuged and the supernatant is used as the working solution of the standard to be tested, the solution of the quality control substance to be tested, and the sample to be tested.

[0072] Table 4 Automated analyte extraction process

[0073] Liquid chromatography-tandem mass spectrometry (LC-MS) conditions Chromatographic conditions: Chromatographic separation was performed on a Kinetex XB-C18 column (2.6 μm, 100 Å, 100 × 2.1 mm) at 40°C with an injection volume of 2 μL. The mobile phase consisted of (A) 0.2% formic acid in water and (B) 0.2% formic acid in acetonitrile. The specific elution gradient is shown in Table 5.

[0074] Table 5. Gradient elution program

[0075] Mass spectrometry conditions: Mass spectrometry detection was performed in multiple reaction monitoring (MRM) mode. Optimized ion source parameters included a source temperature of 550°C, nebulizer and auxiliary heater gas pressures of 60 psi, curtain gas of 40 psi, collision gas of 9 psi, and an ion spray voltage of 4000 V. Precursor and product ions were detected in electrospray positive (ESI+) mode for quantitative analysis. Table 6 shows the optimized ion transitions and parameters, including both quantitative and qualitative analysis.

[0076] Table 6 MRM parameters

[0077] Precursor and product ions of polymyxins E1 and E2 were detected by electrospray ionization (ESI) in positive ion mode. Mass spectrometry / mass spectrometry (MS / MS) settings were optimized to provide the most stable and intense product ions for multiple reaction monitoring (MRM) analysis. The polymyxin E1 precursor ion (Q1) was detected at m / z 390.900, and two distinct product ions (Q3) were selected: m / z 385.000 (a highly abundant fragment, the primary quantification ion) and m / z 101.100 (a confirming qualifier ion). The polymyxin E2 precursor ion (Q1) was detected at m / z 386.100, and the following product ions (Q3) were selected: the primary quantification ion was m / z 380.100, and the qualifier was m / z 101.100. Polymyxin B1 was chosen as the internal standard (IS) due to structural similarity, cost-effectiveness, and minimal clinical interference. The transition of the IS was monitored at m / z 602.569→241.1, and the dwell time for all MRM transitions was 50 ms.

[0078] Detection and Analysis The prepared working solution of the standard to be tested was injected into liquid chromatography-tandem mass spectrometry for analysis. The concentration ratio of the standard to the internal standard was plotted on the X-axis and the peak area ratio was plotted on the Y-axis. Linear regression analysis was performed to obtain the standard curve and the standard curve equation.

[0079] The prepared test sample is injected into liquid chromatography-tandem mass spectrometry for detection and analysis, and the polymyxin E1 and polymyxin E2 in the test plasma sample are quantified based on the obtained standard curve and the standard curve equation.

[0080] The quality control solution to be tested is injected into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the obtained standard curve and standard curve equation, the amount of polymyxin E1 and polymyxin E2 in the quality control solution to be tested is calculated, and the accuracy and precision are calculated based on the theoretical content of polymyxin E1 and polymyxin E2 in the quality control solution to be tested. When the accuracy and precision deviations are within the predetermined acceptance criteria, the test results of the plasma sample to be tested are valid; if the accuracy and precision deviations exceed the predetermined acceptance criteria, the test is repeated.

[0081] Methodological validation According to the guidelines for bioanalytical method validation, the analytical method was validated for specificity, linearity, lower limit of detection (LLOQ), precision, accuracy, recovery, matrix effect, and stability.

[0082] Specificity and selectivity By comparing the blank plasma chromatogram with the chromatogram of the minimum detection limit sample, evaluate whether there is interference between the chromatographic peaks of polymyxin E1, E2, and polymyxin B1 in plasma. For polymyxin E1 and E2, the interference signal response in the blank sample should not exceed 20% of the minimum detection limit, and for the internal standard, it should not exceed 5%. Figure 3 、 Figure 4 、 Figure 5 As shown in Figure 2, there are no interfering peaks from endogenous chemicals in plasma within the retention times of polymyxins E1 and E2, as well as polymyxin B1, which means that their separation and quantification are not affected. The retention times of polymyxins E1, E2, and polymyxin B1 in plasma are 3.42, 3.22, and 3.58 minutes, respectively (Figure 2). Figure 6 、 Figure 7 、 Figure 8 There was no significant interference in the blank plasma chromatogram within the retention time of the analytes and polymyxin B1 (as shown in Figure 3 、 Figure 4 、 Figure 5 Typical chromatograms of analytes from patient plasma are shown in Figure 2. Figure 12 、 Figure 13 、 Figure 14 shown.

[0083] Linearity and lower limit of detection (LLOQ) The linear range for measuring polymyxin E1 and E2 was determined based on eight standard curve working solutions at clinical treatment intervals; linear regression analysis was performed by the least squares method with a weighting factor of 1 / x². The standard curve regression equations for polymyxin E1 and E2 were y = 9.55x + 0.0842 (r = 0.9994) and y = 8.21x - 0.0036 (r = 0.9997), respectively. Polymyxin E1 showed significant linearity in the concentration range of 0.01-20 μg / mL. Polymyxin E2 also showed significant linearity in the concentration range of 0.01-20 μg / mL. The method met the bioanalytical standards and achieved a lower limit of detection (LLOQ) of 0.01 μg / mL for polymyxin E1 and E2 (such as Figure 6 、 Figure 7 、 Figure 8 These assays demonstrate increased sensitivity compared to conventional methods, allowing for accurate quantification of low concentrations of colistin in clinical samples.

[0084] Accuracy and precision The accuracy of the method was determined by the percentage difference between the average measured concentration and the theoretical concentration of the quality control samples. Intra-day accuracy and precision were determined by measuring six replicates of three quality control concentrations (low, medium, and high). The study was repeated over six days to measure inter-day accuracy and precision. Precision was expressed as the relative standard deviation (RSD) between replicate measurements of each quality control sample. Accuracy was expressed as the deviation between the calculated concentration and the nominal concentration of each quality control sample. The concentration of each sample was determined using a calibration curve constructed that day, and the results are shown in Table 7.

[0085] Table 7 Inter-day and intra-day precision and accuracy of polymyxin E1 and polymyxin E2 in plasma

[0086] Table 7 lists the intra-day and inter-day precision and accuracy data at the four quality control levels. For polymyxin E1 and E2, the intra-day accuracy ranged from -6.97% to 8.20% and -3.60% to 8.52%, respectively, with RSDs for intra-day precision ranging from 3.26% to 9.46% and 2.28% to 7.28%, respectively. For polymyxin E1 and E2, the inter-day accuracy ranged from -1.0% to 3.12% and 1.26% to 3.89%, respectively, with RSDs for inter-day precision ranging from 5.16% to 8.13% and 3.89% to 7.89%, respectively. The method met the predefined acceptance criteria (bias within ±15% and RSD less than 15%), demonstrating its reliability and repeatability for measuring polymyxin E in plasma at clinically relevant concentrations.

[0087] Extraction recovery and matrix effect Extraction recoveries were assessed by spiking blank plasma with polymyxins E1 and E2 at low, medium, and high quality concentrations (LQC, MQC, and HQC), followed by automated magnetic bead extraction. Six replicate samples were prepared for each concentration in three independent batches. Recovery (%) was calculated as follows: Recovery (%) = theoretical concentration / extracted sample concentration × 100%. Matrix-matched standards were prepared by spiking blank plasma with analytes and internal standards (without extraction). Acceptance criteria for recovery were 85–115%, in accordance with bioanalytical method validation guidelines.

[0088] The matrix effect was evaluated by comparing the peak area ratios of polymyxin E1 and E2 added to blank plasma and polymyxin E1 and E2 added to pure solvent at the same concentration. The matrix effect (ME) was calculated as follows: Peak area ratio of pure solvent sample / peak area ratio of matrix-matched sample × 100%. Matrix effect (ME) in the range of 85-115% indicates acceptable matrix interference.

[0089] The results of extraction recovery and matrix effect are shown in Table 8.

[0090] Table 8 Recovery and matrix effect (n=18)

[0091] Table 8 shows the matrix effect and recovery data. At the three quality control concentration levels, the recoveries of polymyxin E1 were 102.3 ± 4.23, 99.91 ± 5.45, and 103.1 ± 8.90%, and those of polymyxin E2 were 101.3 ± 6.10, 101.4 ± 5.81, and 103.3 ± 8.81%. These results demonstrate that the magnetic bead-based extraction method provides satisfactory extraction efficiency without significant interference from endogenous compounds. At the three quality control concentration levels, the matrix effects for polymyxin E1 were 98.99 ± 9.00, 97.98 ± 6.75, and 98.27 ± 1.93%, and for polymyxin E2 were 103.62 ± 12.31, 100.44 ± 7.24, and 99.69 ± 1.94%. This is attributed to the selective binding of the magnetic beads, which effectively removes nonspecifically bound substances and reduces matrix effects.

[0092] Carryover effect Carryover refers to the presence of analytes from the previous run in the LC-MS system and their detection in the next measurement. When a blank sample was injected after a chromatographic run of a high-concentration sample (20 μg / mL for both polymyxins E1 and E2), the chromatogram showed a signal less than 20% of the lower limit of detection (LLOQ) at the peak times of polymyxins E1 and E2, or less than 5% at the peak time of polymyxin B1. Figure 9 、 Figure 10 、 Figure 11 As shown in the Figure 2, no carryover effect was observed for polymyxins E1, E2, and B1. Therefore, it is considered that the carryover effect of polymyxins E1, E2, and B1 during the chromatographic determination is negligible.

[0093] stability To simulate the entire process of sample collection, processing, and storage, which is very similar to routine clinical testing, the stability of polymyxins E1 and E2 was tested. Three replicate samples were tested at two concentrations (0.2 μg / mL and 2 μg / mL) at different temperatures (4°C, room temperature, -20°C, and -80°C) and during freeze-thaw cycles. Thawing occurred at room temperature; freeze-thaw cycles consisted of freezing at -80°C and then thawing at room temperature, repeated three times. The results are shown in Table 9.

[0094] Table 9 Stability test data

[0095] As shown in Table 9, the accuracy of plasma samples remained between 100 ± 15% at all levels, and the method had good stability.

[0096] Compare apps The pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution prepared by protein precipitation method were used to prepare the standard working solution to be tested, the quality control solution to be tested, and the sample to be tested that were pretreated by protein precipitation method; the pretreatment scheme of protein precipitation method is as follows: Transfer 20 μL of the pretreatment standard working solution, pretreatment quality control solution, or plasma sample pretreatment solution to a 1.5 mL polypropylene tube. Add 20 μL of deionized water, 10 μL of the internal standard working solution, and 100 μL of isopropanol to each tube. Vortex the mixture briefly for 10 s and then centrifuge at 13,000 g for 5 minutes at 4°C. Dilute the supernatant fivefold with deionized water. Finally, inject 5 μL of each prepared sample into the LC-MS system.

[0097] The other solution preparation, chromatography and mass spectrometry conditions of the protein precipitation method are the same as those of the present application.

[0098] Taking the detection concentration of polymyxin E1 at 8ug / ml as an example, the response intensity of the protein precipitation method is about 6500cps, while the response intensity of the method of the present application reaches 2.4e 6 The response intensity of the present invention is 1000 times that of the protein precipitation method, and the matrix effect of the protein precipitation method is too strong (e.g. Figure 15 、 Figure 16 shown).

[0099] Clinical Application This method was successfully used for therapeutic drug monitoring (TDM) applications to determine plasma levels of polymyxin E. Blood samples were collected from ten patients receiving intravenous polymyxin E sodium mesylate every 12 hours, with doses ranging from 50 to 150 mg of polymyxin E per intravenous injection. Steady-state trough and peak concentrations were measured within 30 minutes before the fourth or fifth dose. Two milliliters of blood were collected in purple-capped anticoagulant tubes. After collection, whole blood samples were centrifuged at 3000 g for 10 minutes at 4°C, and the supernatant was saved for further analysis. Polymyxin E concentrations were calculated using a validated LC-MS / MS method as the sum of polymyxin E1 and E2 concentrations. Clinical application is shown in Table 10.

[0100] Table 10 Monitoring of patients receiving polymyxin E therapy

[0101] As shown in Table 10, trough concentrations of polymyxin E in blood samples from ten patients receiving polymyxin E sodium mesylate for injection ranged from 0.21 μg / mL to 7.63 μg / mL, and peak concentrations ranged from 0.57 μg / mL to 9.43 μg / mL. Patient 2 exhibited a higher trough concentration (3.14 μg / mL) in his blood sample, accompanied by an elevated serum creatinine level (166.19 μmol / L), demonstrating a direct correlation between supratherapeutic concentrations of polymyxin E (>2.3 μg / mL) and markers of renal impairment. This further emphasizes the critical role of therapeutic drug monitoring (TDM) in preventing dose-related toxicity while maintaining effectiveness against multidrug-resistant pathogens. This method showed excellent selectivity in LC-MS / MS analysis, with recoveries ranging from 99.91±5.45% to 103.1±8.90%, negligible matrix effects, a detection limit of 0.01 μg / mL, and a linear range of 0.01-20 μg / mL, providing a reliable solution for clinical TDM.

[0102] The present application has been described in detail above. Specific examples have been used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only intended to help understand the present disclosure and its core ideas. It should be noted that, for those skilled in the art, without departing from the principles of this application, various improvements and modifications may be made to this application, and such improvements and modifications also fall within the scope of protection of the claims of this application.

Claims

1. A method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry, wherein the method uses polymyxin B1 as an internal standard, characterized in that: The method comprises: pre-treating a plasma sample with HLB magnetic beads to obtain a sample to be tested; then quantifying the content of polymyxin E in the sample to be tested using liquid chromatography-tandem mass spectrometry; and activating the HLB magnetic beads with methanol before pre-treating the plasma sample with the HLB magnetic beads.

2. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The step of pre-treating the plasma sample using HLB magnetic beads to obtain the sample to be tested includes: S1': plasma sample pretreatment: take a plasma sample, add the internal standard working solution and diluent, vortex mix, and obtain the plasma sample pretreatment solution; S2': Automated extraction of plasma sample pretreatment solution; add the plasma sample pretreatment solution obtained in step S1' to the magnetic bead extraction plate preloaded with activated HLB magnetic beads, and perform magnetic bead activation, equilibrium, adsorption, washing and elution steps in sequence through the magnetic bead extractor, and collect the eluate as the sample to be tested.

3. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: In step S2', the eluent is a 5% formic acid-30% acetonitrile aqueous solution.

4. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The liquid chromatography conditions are as follows: a Kinetex XB-C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 2.6 μm; a column temperature of 40°C; an injection volume of 2 μL; mobile phase A consisting of a 0.2% formic acid-water solution; and mobile phase B consisting of a 0.2% formic acid-acetonitrile solution; gradient elution is used to pass the mobile phase through the column; and a flow rate of 0.35 ml / min.

5. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that: The conditions of the gradient elution are as follows: elution time is 0 minute to 0.5 minute, the volume ratio of mobile phase A: mobile phase B is 96:4; elution time is 0.5 minute to 2 minutes, the volume ratio of mobile phase A: mobile phase B changes from 96:04 to 82:18; elution time is 2 minutes to 3.5 minutes, the volume ratio of mobile phase A: mobile phase B changes from 82:18 to 77:23; elution time is 3.5 minutes to 3.6 minutes, the volume ratio of mobile phase A: mobile phase B changes from 77:23 to 10:90; elution time is 3.6 minutes to 4.6 minutes, the volume ratio of mobile phase A: mobile phase B is maintained at 10:90; elution time is 4.6 minutes to 4.7 minutes, the volume ratio of mobile phase A: mobile phase B changes from 10:90 to 96:4; elution time is 4.7 minutes to 5.5 minutes, and the volume ratio of mobile phase A: mobile phase B is maintained at 96:

4.

6. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometer adopts electrospray ionization positive ion mode to detect the precursor ions and product ions of polymyxin E1, polymyxin E2 and polymyxin B1 to perform quantitative analysis of polymyxin E1 and polymyxin E2.

7. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: The quantitative ion pair of polymyxin E1 is 390.900 and 385.000; the qualitative ion pair of polymyxin E1 is 390.900 and 101.100; the quantitative ion pair of polymyxin E2 is 386.100 and 380.100; the qualitative ion pair of polymyxin E2 is 386.100 and 101.100; and the quantitative ion pair of polymyxin B1 is 602.569 and 241.

100.

8. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The method for activating the HLB magnetic beads with methanol is as follows: taking HLB magnetic beads, adding methanol, wherein the mass volume ratio of the HLB magnetic beads to methanol is 5 mg / ml, vortexing and mixing to obtain an activated magnetic bead solution.

9. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: The steps include: S1: Prepare a series of concentrations of standard working solutions, quality control solutions, and internal standard working solutions; the series of concentrations of standard working solutions are prepared by using blank plasma to prepare a series of concentrations of mixed standard working solutions of polymyxin E1 and polymyxin E2, with a concentration range of 0.01-20 μg / mL; the series of concentrations of quality control solutions are prepared by using blank plasma to prepare low, medium, and high concentrations of mixed quality control solutions of polymyxin E1 and polymyxin E2, with a concentration range of 0.2-8 μg / mL; S2: Pretreatment standard working solution, quality control solution: Take the standard working solution or quality control solution prepared in step S1, add the internal standard working solution and the diluent, and vortex mix to obtain the pretreatment standard working solution or pretreatment quality control solution; S3: Automatically extract the pretreatment standard working solution and pretreatment quality control solution prepared in step S2 and the plasma sample pretreatment solution prepared in step S1' to obtain the test standard working solution, test quality control solution, and test sample; S4: injecting the working solution of the standard sample to be tested prepared in step S3 into liquid chromatography-tandem mass spectrometry for analysis, performing linear regression analysis with the concentration ratio of the standard sample to the internal standard as the X-axis and the peak area ratio as the Y-axis to obtain a standard curve and a standard curve equation; S5: injecting the test sample prepared in step S3 into liquid chromatography-tandem mass spectrometry for analysis, and quantifying polymyxin E1 and polymyxin E2 in the test sample based on the standard curve and standard curve equation obtained in step S4; S6: Inject the quality control solution to be tested in step S3 into liquid chromatography-tandem mass spectrometry for analysis. Calculate the amount of polymyxin E1 and polymyxin E2 in the quality control solution to be tested based on the standard curve and standard curve equation obtained in step S4, and calculate the accuracy and precision based on the theoretical content of polymyxin E1 and polymyxin E2 in the quality control solution to be tested. When the accuracy and precision deviations are within the predetermined acceptance criteria, the test result of step S5 is valid; if the accuracy and precision deviations exceed the predetermined acceptance criteria, retest according to steps S1 to S6.

10. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry according to claim 9, characterized in that: The predetermined acceptance criteria described in step S6 are that the deviation is within ±15% and the RSD is less than 15%.

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