A method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry

Abstract

This application provides a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method uses polymyxin B1 as an internal standard, first pretreating the plasma sample with HLB magnetic beads, and then quantifying the polymyxin E content in the plasma using LC-MS / MS. By combining HLB magnetic beads with an automated processing system, this application simplifies the equilibration, extraction, and washing steps, significantly reducing time and material costs, making plasma sample pretreatment simple and rapid, and maximizing the removal of interfering substances. The method exhibits excellent selectivity and specificity, with an average recovery rate between 99.91±5.45% and 103.1±8.90%, negligible matrix effects, a limit of detection (LLOD) of 0.01 μg / mL, and a wide linear range of 0.01–20 μg / mL, providing a rapid solution for clinical monitoring of polymyxin E therapeutics.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Polymyxin E is a cationic peptide antibiotic produced by a subspecies of Bacillus called polymyxin. As a cationic polypeptide, polymyxin E targets the outer membrane of Gram-negative bacteria, disrupting 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 a last resort for treating multidrug-resistant Gram-negative bacterial infections. The main components of polymyxin E are polymyxin E1 and polymyxin E2. Despite its clinical value, its narrow therapeutic window, nephrotoxicity, and neurotoxicity are the main dose-limiting effects. The target maximum value for the 24-hour steady-state plasma concentration-time area under the curve (AUCss, 24h) of polymyxin is 50 mg·h·L. -1 This corresponds to an average steady-state plasma concentration (Css, avg) of 2 μg / mL. Polymyxins have a narrow therapeutic window, and their efficacy and toxicity are closely related to plasma concentrations. Therefore, accurate therapeutic drug monitoring (TDM) is crucial for reducing toxicity risks and preventing antibiotic resistance. Accurate and efficient quantification of polymyxin E in plasma is essential for guiding individualized treatment plans and improving patient outcomes.

[0003] Traditional methods for quantifying polymyxin E, 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 polymyxin E in clinical samples, leading to inaccurate measurements and poor dosage decisions.

[0004] In recent years, liquid chromatography-tandem mass spectrometry (LC-MS) has become a powerful tool for quantifying plasma polymyxin E 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 polymyxin E, the ideal sample pretreatment method should be simple, versatile, and rapid, and should remove interfering substances to the greatest extent possible. Recent LC-MS sample preparation often involves complex pretreatment processes, such as protein precipitation, solid-phase extraction (SPE), or ultrafiltration combined with precipitation. These methods 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, and a specific, rapid, highly sensitive, accurate, and highly stable quantitative analysis method to quantify polymyxin E in plasma, so as to provide timely blood concentration data of polymyxin E for clinical use in order to adjust drug dosage. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry.

[0007] This 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, the plasma sample is pretreated with HLB magnetic beads to obtain the sample to be tested. Then, liquid chromatography-tandem mass spectrometry is used to quantify the content of polymyxin E in the plasma sample. Before pretreatment of the plasma sample with the HLB magnetic beads, the HLB magnetic beads are activated with methanol.

[0008] The process of activating the HLB magnetic beads with methanol involves dispersing the HLB magnetic beads in methanol, wherein the mass-to-volume ratio of the HLB magnetic beads to the methanol is 5 mg / ml, and vortexing to obtain the activated magnetic bead solution.

[0009] 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, vortex to mix and obtain magnetic bead solution, rotation speed 2000 rpm, vortex time 30 s; thus obtaining the activated magnetic bead solution.

[0010] Activated high-load HLB magnetic beads (2 mg / sample) can improve the adsorption efficiency of low-concentration polymyxin E.

[0011] 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 high efficiency of solid-phase extraction, enabling automated, high-throughput capture and purification of trace small molecules (such as hormones, drugs, and pesticides) of moderate or low polarity from complex biological, environmental, or food samples, followed by direct LC-MS / MS analysis. This application utilizes HLB magnetic beads, which consist of a polystyrene matrix coated with an Fe3O4 magnetic core, with surface modifications of phenyl and pyrrolidone functional groups. The HLB magnetic beads of this application integrate hydrophilic pyrrolidone functional groups containing polar amide bonds (-CON-), which can form strong hydrogen bonds with the carboxyl (-COOH), amino (-NH2), or peptide (-NH-CO-) groups of polymyxin E, enhancing hydrophilic adsorption. The hydrophobic portion of the HLB magnetic beads in this application, consisting of a polystyrene matrix backbone, binds to the fatty acid chains or hydrophobic amino acid side chains of polymyxin E through π-π interactions and van der Waals forces. The phenyl modification in the hydrophobic portion enhances hydrophobicity by interacting with hydrophobic segments of the phenylalanine (Phe) residues or fatty acid chains of polymyxin E through π-π stacking. The HLB magnetic beads used in this application possess both hydrophilic and hydrophobic properties, enabling the dual adsorption of polar and nonpolar molecules, thereby efficiently and selectively enriching target analytes from complex biological samples.

[0012] This application utilizes methanol-activated HLB magnetic beads. Methanol can swell the HLB polymer, increasing its specific surface area, promoting the exposure of HLB hydrophobic groups, and facilitating the binding of hydrophobic peptide fragments. Simultaneously, it can optimize the hydrophilicity of the magnetic bead surface and reduce non-specific adsorption. During the adsorption of polymyxin E, methanol can reduce sample matrix interference and improve peptide recovery. Furthermore, methanol can activate carboxyl groups, enhancing electrostatic / hydrogen bond interactions with peptides.

[0013] The polymyxin E includes polymyxin E1 and polymyxin E2; the content of polymyxin E is the sum of polymyxin E1 and polymyxin E2.

[0014] The steps for pre-treating plasma samples using HLB magnetic beads to extract polymyxin E and polymyxin B1 include:

[0015] S1': Plasma sample pretreatment; Take a plasma sample, add internal standard working solution and diluent, vortex mix to obtain plasma sample pretreatment solution;

[0016] In some embodiments, the volume ratio of the plasma sample, internal standard working solution, and diluent is 5:1:1;

[0017] The internal standard working solution is a 0.1% formic acid aqueous solution of 5 μg / mL polymyxin B1; the diluent is a 2% ammonia aqueous solution.

[0018] Diluting with 2% ammonia can reduce the non-specific binding of polymyxin E to the container / protein, significantly improving the recovery rate. Simultaneous addition of polymyxin B1 as an internal standard to the plasma to be tested, and simultaneous extraction of polymyxin B1 during magnetic bead extraction, can compensate for operational variations. The internal standard is corrected throughout the process, which can significantly improve accuracy and precision.

[0019] In some embodiments, plasma sample pretreatment is performed as follows: 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.

[0020] 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, equilibration, adsorption, rinsing and elution steps are performed sequentially by the magnetic bead extractor, and the eluent is collected as the sample to be tested;

[0021] In some embodiments, the automated extraction of the plasma sample pretreatment solution involves preloading activated magnetic beads into wells 1 and 7 of the magnetic bead extraction plate, preloading ultrapure water into wells 2, 8, 4, and 10, preloading the plasma sample pretreatment solution prepared in step S1' into wells 3 and 9, preloading methanol into wells 5 and 11, and preloading eluent into wells 6 and 12. The magnetic bead extraction instrument sequentially performs the steps of magnetic bead activation, magnetic bead equilibration, sample loading and adsorption, rinsing, washing, and elution. Finally, the eluent from wells 6 and 12 is collected, and the supernatant is obtained by centrifugation to obtain the sample to be tested.

[0022] The specific process is as follows:

[0023] Preparation of S21' 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 of the 1st and 7th columns; an appropriate amount of ultrapure water is added to the wells of the 2nd and 8th columns, and the 4th and 10th columns; an appropriate amount of methanol is added to the wells of the 5th and 11th columns; an appropriate amount of elution buffer is added to the wells of the 6th and 12th columns; and the plasma sample pretreatment solution obtained in step S1' is added to the wells of the 3rd and 9th columns.

[0024] The eluent is a 5% formic acid-30% acetonitrile aqueous solution;

[0025] Using a 5% formic acid-30% acetonitrile aqueous solution as the eluent improves elution efficiency. The 5% formic acid reduces the affinity of the target analyte for HLB magnetic beads through protonation, promoting desorption. Furthermore, the 5% formic acid adjusts the pH to acidic, inhibiting ionization of the target analyte and reducing non-specific binding to residual silanol groups on the magnetic bead surface. The 30% acetonitrile disrupts hydrophobic interactions, resulting in effective elution. Acetonitrile's high miscibility with water prevents phase separation, ensuring uniform elution. The 5% formic acid-30% acetonitrile aqueous solution is compatible with subsequent LC-MS / MS analysis; formic acid enhances the signal response in positive ion mode; and the 30% acetonitrile content reduces solvent effects and improves peak shape. Using this solution also reduces matrix interference; formic acid precipitates residual proteins, and acetonitrile reduces phospholipid solubility, with both synergistically reducing mass spectrometry ion suppression effects. A 5% formic acid-30% acetonitrile aqueous solution can be used as an eluent to balance the adsorption and elution of HLB magnetic beads. The 5% formic acid maintains the hydrophilic interaction, while the 30% acetonitrile regulates the hydrophobic elution.

[0026] In some embodiments, the magnetic bead extraction plate is prepared by placing 200 μL of activated magnetic bead solution in wells 1 and 7; adding 400 μL of ultrapure water to wells 2 and 8, and wells 4 and 10; adding 200 μL of methanol to wells 5 and 11; adding 200 μL of elution buffer to wells 6 and 12; and adding an appropriate amount of plasma sample pretreatment solution to wells 3 and 9.

[0027] S22': Automated extraction; Place the prepared magnetic bead extraction plate in the magnetic bead extractor and perform automated extraction according to the following procedure:

[0028] Magnetic bead activation: The magnetic beads in the first and seventh columns oscillate for 30 seconds, are magnetically attracted for 30 seconds, and then transferred to the second and eighth columns;

[0029] Magnetic bead balancing: The magnetic beads in the 2nd and 8th columns oscillate for 60 seconds, are magnetically attracted for 30 seconds, and then transferred to the 3rd and 9th columns;

[0030] Sample loading: The magnetic beads in the 3rd and 9th columns vibrate for 150 seconds, are magnetically attracted for 30 seconds, and then transferred to the 4th and 10th columns;

[0031] Magnetic bead rinsing: The magnetic beads in the 4th and 10th columns oscillate for 60 seconds, are magnetically attracted for 30 seconds, and then transferred to the 5th and 11th columns;

[0032] Magnetic bead cleaning: The magnetic beads in the 5th and 11th columns oscillate for 60 seconds, are magnetically attracted for 30 seconds, and then transferred to the 6th and 12th columns;

[0033] Magnetic bead elution: The magnetic beads in the 6th and 12th wells were oscillated for 150 seconds, magnetically attracted for 30 seconds, and then transferred to the 1st and 7th wells and discarded; the eluent contained the target analyte.

[0034] S23': Centrifuge the eluent from the 6th and 12th wells and take the supernatant of the eluent as the sample to be tested;

[0035] In the preparation and automated extraction process of the magnetic bead extraction plate of this application, the first and seventh wells are filled with activated magnetic bead solution to ensure consistent binding capacity between the magnetic beads and the analyte; the second and eighth wells are filled with ultrapure water for aqueous phase equilibration to remove methanol residue; the fourth and tenth wells are filled with ultrapure water for a weak washing step to remove salts and water-soluble non-specific adsorbed impurities; the fifth and eleventh wells are filled with methanol for a strong washing step to remove phospholipids and hydrophobic interfering substances; the sixth and twelfth wells are filled with acidic elution buffer to selectively release polymyxin E and polymyxin B1, reduce co-elution impurities, and finally elute the target analyte, polymyxin E; in the automated extraction process, the gradient washing design of weak washing (water), strong washing (methanol), and acidic elution, combined with the extraction of HLB magnetic beads with carboxyl-modified methanol activation, synergistically enhances the specificity and selectivity of magnetic beads in extracting polymyxin E from plasma, resulting in good recovery rate and reduced matrix effect.

[0036] The liquid chromatography conditions were as follows: a Kinetex XB-C18 column with a length of 100 mm × inner diameter of 2.1 mm × packing particle size of 2.6 μm or equivalent performance was used; the column temperature was 40 °C; the injection volume was 2 μL; mobile phase A consisted of 0.2% formic acid-water solution; mobile phase B consisted of 0.2% formic acid-acetonitrile solution; gradient elution was used to pass the mobile phase through the column; the flow rate was 0.35 ml / min.

[0037] The gradient elution conditions are as follows: elution time 0 to 0.5 minutes, mobile phase A:mobile phase B volume ratio 96:4; elution time 0.5 to 2 minutes, mobile phase A:mobile phase B volume ratio changes from 96:04 to 82:18; elution time 2 to 3.5 minutes, mobile phase A:mobile phase B volume ratio changes from 82:18 to 77:23; elution time 3.5 to 3.6 minutes, mobile phase A:mobile phase B volume ratio changes from 77:23 to 10:90; elution time 3.6 to 4.6 minutes, mobile phase A:mobile phase B volume ratio remains at 10:90; elution time 4.6 to 4.7 minutes, mobile phase A:mobile phase B volume ratio changes from 10:90 to 96:4; elution time 4.7 to 5.5 minutes, mobile phase A:mobile phase B volume ratio remains at 96:4.

[0038] Under the aforementioned liquid chromatography conditions, polymyxin E1 and E2 are separated from the internal standard polymyxin B1 by retention time, achieving effective chromatographic peak separation; post-column washing for 1.2 min ensures thorough elution, removes residues, and reduces carryover effect.

[0039] The mass spectrometry uses electrospray ionization positive ion mode to detect the precursor ions and product ions of polymyxin E1, polymyxin E2 and polymyxin B1 for quantitative analysis of polymyxin E1 and polymyxin E2.

[0040] In some embodiments, the mass spectrometry parameters are as follows: ion source temperature is 550°C, nebulizing gas and auxiliary heating gas are both 60 psi, curtain gas is 40 psi, collision gas is 9 psi, and ion spray voltage is 4000 V; the 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.

[0041] The quantitative ion pairs of polymyxin E1 are 390.900 and 385.000; the qualitative ion pairs of polymyxin E1 are 390.900 and 101.100; the quantitative ion pairs of polymyxin E2 are 386.100 and 380.100; the qualitative ion pairs of polymyxin E2 are 386.100 and 101.100; the quantitative ion pairs of polymyxin B1 are 602.569 and 241.1; wherein, the polymyxin... The precursor ion of E1 is the ion at 390.900; the product ions of polymyxin E1 are the ions at 385.000 and 101.100; the precursor ion of polymyxin E2 is the ion at 386.100; the product ions of polymyxin E2 are the ions at 380.100 and 101.100; the precursor ion of polymyxin B1 is the ion at 602.569; the product ion of polymyxin E2 is the ion at 241.1.

[0042] This application selects 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 from plasma and achieve specificity for the detection of polymyxin E1 and E2; m / z 385.00 and 380.1 are high-abundance quantitative ions with less interference, which can improve detection sensitivity; m / z 101.1 helps to confirm the identity of the target analyte; the internal standard (polymyxin B1, Q1:602.569→Q3:241.1) corrects for matrix effects and reduces matrix effects; dual-charge ion monitoring enhances signal linearity.

[0043] The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry includes the following steps:

[0044] S1: Prepare standard working solutions, quality control solutions, and internal standard working solutions of various concentrations;

[0045] The series of standard working solutions are prepared using blank plasma to create a series of mixed standard working solutions of polymyxin E1 and polymyxin E2, with a concentration range of 0.01~20μg / mL.

[0046] The series of quality control solutions are prepared using blank plasma to create mixed quality control solutions of polymyxin E1 and polymyxin E2 at low, medium, and high concentrations, with a concentration range of 0.2~8 μg / mL.

[0047] In some embodiments, the concentration of the polymyxin E1 and polymyxin E2 mixed standard working solution includes 20.00, 10.00, 5.00, 2.00, 0.50, 0.10, 0.05, and 0.01 μg / mL; the concentration of the polymyxin E1 and polymyxin E2 mixed quality control solution includes 0.2, 1, and 8 μg / mL.

[0048] The internal standard working solution is a polymyxin B1 solution with a concentration of 5 μg / mL prepared using 0.1% formic acid water;

[0049] 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 a good linear relationship.

[0050] S2: Pretreatment standard working solution and quality control solution: Take the standard working solution or quality control solution obtained in step S1, add the internal standard working solution and diluent, 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;

[0051] In some embodiments, the pretreatment of the standard working solution or the quality control solution is as follows: take 100 μL of standard solution or quality control solution, add 20 μL of internal standard working solution and 100 μL of diluent, vortex mix well to obtain the pretreated standard working solution or the pretreated quality control solution.

[0052] S3: Automated extraction of the pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution obtained in step S2 and step S1' to obtain the test standard working solution, test quality control solution, and test sample; the automated extraction of the pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution obtained in step S2 and step S1' to obtain the test standard working solution, test quality control solution, and test sample is based on the automated extraction of the plasma sample pretreatment solution described in step S2', and the pre-filled wells in the 3rd and 9th columns are either the pretreatment standard working solution or the pretreatment quality control solution obtained in step S2 or the plasma sample pretreatment solution obtained in step S1';

[0053] S4: Inject the working solution of the standard to be tested prepared in step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Use the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio as the Y-axis to perform linear regression analysis to obtain the standard curve and the standard curve equation.

[0054] S5: Inject the sample to be tested obtained in step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the standard curve and standard curve equation obtained in step S4, quantify polymyxin E1 and polymyxin E2 in the plasma sample to be tested.

[0055] S6: Inject the test quality control solution from step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the standard curve and standard curve equation obtained in step S4, calculate the amounts of polymyxin E1 and polymyxin E2 in the test quality control solution. Compare the accuracy and precision with the theoretical contents of polymyxin E1 and polymyxin E2 in the test quality control solution. If the deviation of accuracy and precision is within the predetermined acceptance standard, the detection result of step S5 is valid. If the deviation of accuracy and precision exceeds the predetermined acceptance standard, re-detect according to steps S1 to S6.

[0056] The predetermined acceptance criteria are a deviation within ±15% and an RSD of less than 15%.

[0057] This application ensures the accuracy and reliability of polymyxin E monitoring data through internal standard calibration and full-process monitoring of quality control materials.

[0058] In summary, this application includes the following beneficial technical effects:

[0059] 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 makes plasma sample pretreatment simple and rapid, and maximizes the removal of interfering substances. Combined with optimized LC-MS parameters, this method exhibits excellent selectivity and specificity in LC-MS analysis, with average recoveries ranging from 99.91±5.45% to 103.1±8.90%, and negligible matrix effects. The 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 clinical therapeutic monitoring of polymyxin E.

[0060] 2. The technical features of this application have a synergistic effect. The HLB magnetic beads, MRM specific ion pairs, and gradient elution technology of liquid chromatography used in this application achieve the specificity of the method for detecting polymyxin E. This application uses blank plasma to prepare standard working solutions to offset the matrix differences in magnetic bead extraction and ionization efficiency, and combined with dual-charge ion monitoring, achieves a wide linear range and good linearity. This application uses HLB magnetic bead extraction, 2% ammonia water as a diluent, and high-abundance fragment ions to achieve high detection sensitivity. This application uses automated extraction, internal standard correction, and full-process monitoring of quality control materials, giving the method good precision and accuracy. This application uses methanol to activate HLB magnetic beads, and uses gradient elution during extraction, with simultaneous polymyxin B1 correction ion inhibition / enhancement effect, giving the method good recovery rate and low matrix effect. In chromatographic separation, post-column washing removes residues; there is little carryover effect, and the sample detection stability is good. Compared to commercial SPE columns (Oasis® HLB, 35 RMB / sample), this method reduces the cost per sample by 71% (10 RMB / sample) and increases throughput, making it more suitable for large-scale clinical laboratory applications. Attached Figure Description

[0061] The present application will be described in further detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will understand 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 this disclosure. Furthermore, unless specifically indicated, the drawings are only schematic representations of the composition or structure of the described objects and may contain exaggerated depictions, and the drawings are not necessarily drawn to scale.

[0062] Figure 1 Structural diagrams of polymyxin E1, polymyxin E2, and polymyxin B1; among which, Figure 1The first part of the formula is a schematic diagram of the structure of polymyxin E1; Figure 1 Part II of the formula is a schematic diagram of the structure of polymyxin E2; Figure 1 Formula III is a schematic diagram of the structure of polymyxin B1.

[0063] Figure 2 The flowchart shows the magnetic bead extraction process, where A is a schematic diagram of a 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.

[0064] Figure 3 MRM chromatogram of polymyxin E1 in a blank plasma sample without polymyxin B1.

[0065] Figure 4 MRM chromatogram of polymyxin E2 in a blank plasma sample without polymyxin B1.

[0066] Figure 5 MRM chromatogram of polymyxin B1 in a blank plasma sample without added polymyxin B1.

[0067] Figure 6 MRM chromatogram of polymyxin E1 in plasma samples with added 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantitation).

[0068] Figure 7 MRM chromatogram of polymyxin E2 in plasma samples with added 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantification).

[0069] Figure 8 MRM chromatogram of polymyxin B1 in plasma samples with added 0.01 μg / mL polymyxin E1 and 0.01 μg / mL polymyxin E2 (lower limit of quantitation).

[0070] Figure 9 Chromatogram of polymyxin E1 for carrier effect detection.

[0071] Figure 10 Chromatogram of polymyxin E2 for carrier effect detection.

[0072] Figure 11 Chromatogram of polymyxin B1 for carrier effect detection.

[0073] Figure 12 Chromatogram of polymyxin E1 in a patient's plasma sample.

[0074] Figure 13 Chromatogram of polymyxin E2 in a patient's plasma sample.

[0075] Figure 14 Chromatogram of polymyxin B1 in a patient's plasma sample.

[0076] Figure 15 Chromatogram of polymyxin E1 after protein precipitation pretreatment.

[0077] Figure 16 This is the chromatogram of polymyxin E1 after magnetic bead pretreatment in this application. Detailed Implementation

[0078] The following is in conjunction with the appendix Figures 1 to 16 This disclosure will be explained in detail.

[0079] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0080] Chemicals and reagents

[0081] Polymyxin E1 sulfate standard, CAS No. 1066-17-7, purchased from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. in Nanjing, China, batch number 20211118-1, purity 78.4%, structural formula as follows. Figure 1 As shown in Equation I.

[0082] Polymyxin sulfate E2 standard, CAS No. 30387-41-8, purchased from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. in Nanjing, China, batch number 20221203, purity 79.9%, structural formula as follows: Figure 1 As shown in Equation II.

[0083] Internal standard compound (IS): Polymyxin B1 standard, CAS No. 4135-11-9, purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China, catalog number P343277-1mg, batch number F2418155, structural formula as follows Figure 1 As shown in Equation III.

[0084] Depend on Figure 1 It is known 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.

[0085] Magnetic beads: HLB magnetic beads (30μm) are composed of Fe3O4 magnetic cores coated with polystyrene matrix and modified with phenyl and pyrrolidone functional groups on the surface. The specific surface area is 680 m² / g and the average pore size is 80 Å. They were purchased from Junrong Biotechnology Co., Ltd. in Hangzhou, China.

[0086] HPLC-grade methanol, acetonitrile, and formic acid were purchased from Fisher Chemical Company, Fairlaun, New Jersey, USA.

[0087] Ultrapure water was prepared using the Milli-Q water purification system from Merck Millipore in Darmstadt, Germany.

[0088] Unless otherwise specified, all other reagents used in the embodiments of this application are from conventional commercially available products.

[0089] Instruments and equipment

[0090] LC-MS: AB Sciex Triple Quad™ 4500 mass spectrometer and Jasper™ high performance liquid chromatography system. Data acquisition and statistical analysis were performed using Analyst 1.6.3 software (SCIEX, USA).

[0091] Magnetic bead extractor: Smart 32, purchased from Guangzhou Da'an Biotechnology Co., Ltd., China.

[0092] Solution preparation

[0093] Accurately weigh the polymyxin E1 and polymyxin E2 standards, and prepare a stock solution using a 0.1% formic acid-10% methanol aqueous solution. Then, dilute with a 0.1% formic acid aqueous solution to prepare a mixed standard stock solution with a concentration ranging from 10 to 1000 μg / mL. Next, use blank plasma for a series of dilutions 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 samples. The specific preparation process for the standard working solutions and quality control samples is as follows:

[0094] Preparation of standard mother liquor

[0095] Accurately weigh 4 mg of polymyxin E1 sulfate standard, add 1568 μL (2 ml * 0.784, the conversion coefficient between the purity of polymyxin E1 sulfate standard and the mass of sulfate is 0.784) of 0.1% formic acid-10% methanol aqueous solution to dissolve it, and obtain a 2 mg / ml polymyxin E1 standard stock solution.

[0096] Accurately weigh 4 mg of polymyxin E2 sulfate standard, add 1598 μL (2 ml * 0.799, the conversion factor between the purity of polymyxin E2 sulfate standard and the mass of sulfate is 0.799) of 0.1% formic acid-10% methanol aqueous solution to dissolve it, and obtain a 2 mg / ml polymyxin E2 standard stock solution.

[0097] Accurately weigh 2 mg of polymyxin B1 standard and dissolve it in 1900 μL (2 ml * 0.95, with a purity conversion factor of 0.95) of 0.1% formic acid-10% methanol aqueous solution to obtain a 1 mg / ml polymyxin B1 standard stock solution.

[0098] Preparation of mixed standard stock solution

[0099] Take the stock solution of polymyxin E1 standard and the stock solution of polymyxin E2 standard, and prepare them according to Table 1.

[0100] Table 1. Preparation of Mixed Standard Stock Solution

[0101]

[0102] Preparation of standard working solutions and quality control products

[0103] The standard working solutions and quality control samples were prepared in the same plasma matrix as the test samples. The 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. The concentrations of the quality control polymyxin E1 and E2 were 0.2, 1, and 8 μg / mL, respectively. The standard working solutions and quality control samples were prepared according to Table 2.

[0104] Table 2 Preparation of Standard Working Solutions and Quality Control Samples

[0105]

[0106] Preparation of internal standard working solution

[0107] Take the stock solution of polymyxin B1 standard and then prepare the internal standard working solution according to Table 3.

[0108] Table 3 Preparation of Internal Standard Working Solution

[0109]

[0110] All final concentrations of the solutions are expressed in μg / mL of free alkali. Standard stock solutions, mixed standard stock solutions, standard curve working solutions, and quality control samples were all stored at -80℃.

[0111] Preparation of diluent (2% ammonia solution)

[0112] Place 49ml of pure water into a 50ml centrifuge tube, add 1ml of ammonia water and vortex to mix to obtain the diluted solution (2% ammonia water).

[0113] Preparation of eluent (5% formic acid - 30% acetonitrile water)

[0114] Take 2.5 ml formic acid, 15 ml acetonitrile, and 35 ml pure water and place them in a 50 ml centrifuge tube. Vortex to mix to obtain the elution buffer (5% formic acid - 30% acetonitrile water).

[0115] Preparation of activated magnetic bead liquid

[0116] Accurately weigh 2 mg of HLB magnetic beads, add 400 μL of methanol, vortex to mix, and obtain activated magnetic bead solution. Rotate at 2000 rpm for 30 s.

[0117] HLB magnetic beads were used for the pretreatment of standard working solutions, quality control solutions, and plasma samples.

[0118] The pretreatment of standard working solutions, quality control solutions, and plasma samples using HLB magnetic beads involves first pretreating the standard working solutions, quality control solutions, and plasma samples to obtain pretreated standard working solutions, pretreated quality control solutions, and pretreated plasma sample solutions; then, automated extraction is performed using an HLB magnetic bead analyzer to obtain the standard working solutions, quality control solutions, and samples to be tested. The specific pretreatment process is as follows:

[0119] Preparation of pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution

[0120] Take 100 μL of standard working solution, add 20 μL of internal standard working solution and 100 μL of diluent, vortex to mix, and the pretreated standard working solution is obtained.

[0121] Take 100 μL of the quality control solution, add 20 μL of internal standard working solution and 100 μL of diluent, vortex to mix, and the pretreated quality control solution is obtained.

[0122] Take 100 μL of the plasma to be tested, add 20 μL of internal standard working solution and 100 μL of diluent, vortex to mix, and the plasma sample pretreatment solution is obtained.

[0123] Preparation of the working solution of the standard to be tested, the quality control solution to be tested, and the sample to be tested.

[0124] The preparation of standard working solutions, quality control samples, and test samples was carried out using HLB magnetic beads and automated extraction using a magnetic bead extractor. Activated magnetic bead solution was pre-loaded into wells 1 and 7; ultrapure water into wells 2, 8, 4, and 10; the working solution of the standard to be tested, the quality control solution, or plasma sample pretreatment solution into wells 3 and 9; methanol into wells 5 and 11; and eluent into wells 6 and 12. The magnetic bead extraction plate was then subjected to the following steps sequentially: magnetic bead activation, magnetic bead equilibration, sample loading and adsorption, rinsing, washing, and elution. Finally, the eluent from wells 6 and 12 was collected, and the supernatant was obtained by centrifugation to obtain the working solution of the standard to be tested, the quality control solution, or the test sample.

[0125] The automated extraction process using HLB magnetic beads and a magnetic bead analyzer is as follows:

[0126] Using a magnetic bead extractor (Smart 32, Guangzhou Da'an Biotechnology Co., Ltd., China), such as... Figure 2 Sample preparation is performed using the device shown in Figure B. The device is equipped with two sets of eight parallel strips and can accommodate two 96-well deep-well plates (such as...). Figure 2 As shown in Figure A), each parallel strip consists of a non-magnetic sheath cap and a magnetic rod. The magnetic rod's extension and retraction achieve the stirring and adsorption of magnetic beads. The device allows for programmable control of the strip's movement between the rows of holes.

[0127] The activated magnetic bead solution (2.0 mg magnetic beads dispersed in 400 μL methanol) was vortexed (2000 rpm, 30 seconds), and 200 μL of the solution was aliquoted into wells 1 and 7 of a 96-well plate, respectively. 400 μL of ultrapure water was added to wells 2 and 8, respectively. 200 μL of methanol was added to wells 5 and 11, respectively. 200 μL of elution buffer (5% formic acid-30% acetonitrile) was added to wells 6 and 12, respectively. 100 μL of pretreatment standard working solution, pretreatment quality control solution, or plasma sample pretreatment solution was added to 20 μL of internal standard working solution and 100 μL of diluent (2% ammonia), vortexed, and then transferred to wells 3 and 9, respectively.

[0128] The fully automated analyte extraction process is as follows (e.g.) Figure 2(As shown in C in Table 4): The sheath cap is lowered to wells 1 and 7, and vertical oscillation is performed for 60 seconds to achieve complete activation and uniform dispersion of the HLB magnetic beads. Then, a magnetic rod is inserted into the sheath cap for magnetization; the beads are adsorbed for 30 seconds and then moved to wells 2 and 8 for aqueous phase equilibration (oscillation for 60 seconds, adsorption for 30 seconds). Subsequently, the beads are moved to wells 3 and 9 to extract the target polymyxin from the sample (oscillation for 150 seconds, adsorption for 30 seconds). The beads are then moved to wells 4 and 10 for weak washing (aqueous phase, oscillation for 60 seconds, adsorption for 30 seconds), and then to wells 5 and 11 for strong washing (methanol phase, oscillation for 60 seconds, adsorption for 30 seconds). Finally, the beads are eluted in wells 6 and 12 by oscillation for 150 seconds to obtain the eluent. The magnetic rod collects the beads for 30 seconds and is then discarded in wells 1 and 7 to complete the process. The eluent contains the target analyte. Centrifuge the eluent and collect the supernatant as the working solution for the standard, the quality control solution, and the sample to be tested.

[0129] Table 4 Automated Analyte Extraction Process

[0130]

[0131] Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) Conditions

[0132] Chromatographic conditions: Chromatographic separation was performed on a Kinetex XB-C18 column (2.6 μm, 100 Å, 100 × 2.1 mm) at a column temperature of 40 °C and an injection volume of 2 μL. The mobile phase consisted of (A) 0.2% formic acid aqueous solution and (B) 0.2% formic acid acetonitrile solution, and the specific elution gradient program is shown in Table 5.

[0133] Table 5. Gradient elution procedure

[0134]

[0135] Mass spectrometry conditions: Mass spectrometry detection was performed in multiple reaction monitoring (MRM) mode. Optimized ion source parameters were: ion source temperature 550℃, nebulizer gas and auxiliary heating gas both 60 psi, curtain gas 40 psi, collision gas 9 psi, and ion spray voltage 4000 V. Precursor and product ions were detected by electrospray positive ion (ESI+) mode for quantitative analysis. Table 6 shows the optimized ion transitions and parameters, including quantitative and qualitative analysis.

[0136] Table 6 MRM Parameters

[0137]

[0138] Precursor and product ions of polymyxin E1 and E2 were detected using 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 precursor ion of polymyxin E1 (Q1) was detected at m / z 390.900, and two distinct product ions (Q3) were selected: m / z 385.000 (high abundance fragment, major quantitative ion) and m / z 101.100 (confirmatory qualitative ion). The precursor ion of polymyxin E2 (Q1) was detected at m / z 386.100, and the following product ion (Q3) was selected: the major quantitative ion at m / z 380.100 and the qualitative ion at m / z 101.100. Due to structural similarity, cost-effectiveness, and minimal clinical interference, polymyxin B1 was selected as the internal standard (IS). IS transitions were monitored at m / z 602.569→241.1, and the dwell time for all MRM transitions was 50 ms.

[0139] Detection and Analysis

[0140] The prepared working solution of the standard to be tested was injected into liquid chromatography-tandem mass spectrometry for detection and analysis. The concentration ratio of the standard to the internal standard was used as the X-axis and the peak area ratio as the Y-axis. Linear regression analysis was performed to obtain the standard curve and the standard curve equation.

[0141] The prepared test sample was injected into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the obtained standard curve and standard curve equation, the polymyxin E1 and polymyxin E2 in the test plasma sample were quantified.

[0142] The test quality control solution is injected into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the obtained standard curve and standard curve equation, the amounts of polymyxin E1 and polymyxin E2 in the test quality control solution are calculated. The accuracy and precision are calculated by comparing these values ​​with the theoretical contents of polymyxin E1 and polymyxin E2 in the test quality control solution. If the deviations in accuracy and precision are within the predetermined acceptance criteria, the test results of the plasma sample are valid. If the deviations in accuracy and precision exceed the predetermined acceptance criteria, the test is repeated.

[0143] Methodological Validation

[0144] In accordance with the guidelines for validation of bioanalytical methods, the analytical methods were validated for specificity, linearity, limit of detection (LLOQ), precision, accuracy, recovery, matrix effect, and stability.

[0145] Specificity and selectivity

[0146] The presence of interference from polymyxin E1, E2, and B1 peaks in plasma is assessed by comparing the chromatograms of blank plasma with those of samples meeting the limit of detection (LOD). For polymyxin E1 and E2, the interference signal response in the blank sample should not exceed 20% of the LOD, while for the internal standard, it should not exceed 5%. Figure 3 , Figure 4 , Figure 5 As shown, no interfering peaks were observed in plasma for endogenous chemicals within the retention times of polymyxins E1 and E2, as well as polymyxin B1, meaning their separation and quantification were unaffected. The retention times of polymyxins E1, E2, and B1 in plasma were 3.42, 3.22, and 3.58 minutes, respectively (e.g., [missing information]). Figure 6 , Figure 7 , Figure 8 (As shown). Within the retention times of the analyte and polymyxin B1, there was no significant interference in the blank plasma chromatogram (e.g., ...). Figure 3 , Figure 4 , Figure 5 (As shown), a typical chromatogram of the analyte from the patient's plasma is as follows: Figure 12 , Figure 13 , Figure 14 As shown.

[0147] Linearity and Limit of Detection (LLOQ)

[0148] Based on eight standard curve working solutions used in clinical treatment intervals, the linear range for measuring polymyxins E1 and E2 was determined. Linear regression analysis was performed using 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 a significant linear relationship in the concentration range of 0.01–20 μg / mL. Polymyxin E2 also showed a significant linear relationship in the same concentration range. This method meets bioanalytical standards and achieves a limit of least detection (LLOQ) of 0.01 μg / mL for the measurement of polymyxins E1 and E2 (e.g., ...). Figure 6 , Figure 7 , Figure 8 (As shown). These methods are more sensitive than traditional methods, allowing for the accurate quantification of low concentrations of polymyxin E in clinical samples.

[0149] Accuracy and precision

[0150] 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 six repeated measurements of samples at 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 repeated measurements for each quality control sample. Accuracy was expressed as the deviation between the calculated concentration and the nominal concentration for each quality control sample. The concentration of each sample was determined using a calibration curve constructed on the same day, and the results are shown in Table 7.

[0151] Table 7. Inter-day and intra-day precision and accuracy of polymyxin E1 and polymyxin E2 determination in plasma.

[0152]

[0153] Table 7 lists the intra-day and inter-day precision and accuracy data at four quality control levels. For polymyxin E1 and E2, the intra-day accuracy ranged from -6.97% to 8.20% and from -3.60% to 8.52%, respectively, and the intra-day precision RSD ranged from 3.26% to 9.46% and from 2.28% to 7.28%, respectively. For polymyxin E1 and E2, the inter-day accuracy ranged from -1.0% to 3.12% and from 1.26% to 3.89%, respectively, and the inter-day precision RSD ranged from 5.16% to 8.13% and from 3.89% to 7.89%, respectively. This method met the predetermined acceptance criteria (deviation within ±15%, RSD less than 15%), demonstrating its reliability and repeatability in measuring polymyxin E in plasma at clinically relevant concentrations.

[0154] Extraction recovery and matrix effect

[0155] Extraction recoveries were evaluated by adding polymyxin E1 and E2 to blank plasma at low, medium, and high concentrations (LQC, MQC, HQC) followed by automated magnetic bead extraction. For each concentration, six replicate samples were prepared in three independent batches. The recovery rate (%) was calculated as: Recovery rate (%) = Theoretical concentration / Post-extraction sample concentration × 100%. Matrix-matched standards were prepared by adding the analyte and internal standard (without extraction) to blank plasma. The acceptance criteria for recovery were 85-115%, according to the bioanalytical method validation guidelines.

[0156] The matrix effect was assessed by comparing the peak area ratios of extracted blank plasma with added polymyxin E1 and E2 with those with the same concentrations of polymyxin E1 and E2 added to pure solvent. The formula for calculating the matrix effect (ME) is as follows:

[0157] The peak area ratio of pure solvent sample / peak area ratio of matrix-matched sample × 100% indicates acceptable matrix interference with matrix effect (ME) in the range of 85-115%.

[0158] The results of extraction recovery and matrix effect are shown in Table 8.

[0159] Table 8 Recovery and matrix effect (n=18)

[0160]

[0161] 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 the recoveries of polymyxin E2 were 101.3±6.10, 101.4±5.81, and 103.3±8.81%. These results indicate 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 effect of polymyxin E1 was 98.99±9.00, 97.98±6.75, and 98.27±1.93%, and the matrix effect of polymyxin E2 was 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 non-specifically bound substances and reduces the matrix effect.

[0162] Carrying effect

[0163] The carryover effect refers to the phenomenon where analytes remain in the LC-MS system from a previous run and are detected in the next measurement. When a blank sample is injected after a chromatographic run with high concentrations (20 μg / mL for both polymyxin E1 and E2), the chromatograms show signals below 20% of the limit of detection (LLOQ) at the peak times of polymyxin E1 and E2, or below 5% at the peak time of polymyxin B1. Results are as follows... Figure 9 , Figure 10 , Figure 11 As shown, no carryover effect was observed for polymyxins E1, E2, and B1. Therefore, the carryover effect of polymyxins E1, E2, and B1 is considered negligible during chromatographic determination.

[0164] stability

[0165] To simulate the entire process of sample collection, processing, and storage, the stability of polymyxins E1 and E2 was tested, which closely resembles routine clinical testing. The stability of three replicate samples was tested at two levels (0.2 μg / mL and 2 μg / mL) at different temperatures (4℃, room temperature, -20℃, and -80℃) and during freeze-thaw cycles. Thawing was performed at room temperature; freeze-thaw cycles involved freezing at -80℃ and then thawing at room temperature, repeated three times. The results are shown in Table 9.

[0166] Table 9 Stability Experiment Data

[0167]

[0168] As shown in Table 9, the accuracy of plasma samples remained within 100 ± 15% at all levels, indicating good stability of the method.

[0169] Comparison Application

[0170] The pretreatment standard working solution, pretreatment quality control solution, and plasma sample pretreatment solution were prepared using the protein precipitation method. These were then used to prepare the target standard working solution, target quality control solution, and target sample after protein precipitation pretreatment. The protein precipitation pretreatment scheme is as follows:

[0171] Transfer 20 μL of 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 internal standard working solution, and 100 μL of isopropanol to each tube. Vortex the mixture briefly for 10 s, then centrifuge at 13000 g for 5 minutes at 4 °C. Dilute the supernatant 5-fold with deionized water. Finally, inject 5 μL of each prepared sample into the LC-MS system.

[0172] The preparation of other solutions, chromatographic and mass spectrometric conditions for the protein precipitation method are the same as those in this application.

[0173] Taking polymyxin E1 at a concentration of 8 μg / ml as an example, comparing the protein precipitation method and the method of this application, the response intensity of the protein precipitation method is approximately 6500 cps, while the response intensity of the method of this application reaches 2.4 e. 6 The response intensity of the method in this application 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 (As shown).

[0174] Clinical Application

[0175] This method was successfully used in therapeutic drug monitoring (TDM) to determine plasma polymyxin E levels. Blood samples were collected from ten patients receiving injectable polymyxin E mesylate sodium, at doses ranging from 50 to 150 mg intravenously every 12 hours. Steady-state trough and peak concentrations were measured 30 minutes before the fourth or fifth dose. Two mL of blood was collected and placed in a purple-capped anticoagulant tube. After collection, whole blood samples were centrifuged at 3000 g for 10 minutes at 4°C, and the supernatant was retained for further analysis. Polymyxin E concentration was calculated using a validated LC-MS / MS method, i.e., the sum of polymyxin E1 and E2 concentrations. Clinical applications are shown in Table 10.

[0176] Table 10 Monitoring of Polymyxin E Treatment in Patients

[0177]

[0178] Table 10 shows that in the blood samples of ten patients treated with injectable polymyxin E mesylate, the trough concentration of polymyxin E ranged from 0.21 μg / mL to 7.63 μg / mL, and the peak concentration ranged from 0.57 μg / mL to 9.43 μg / mL. Patient 2 showed a higher trough concentration (3.14 μg / mL) in their blood sample, along with elevated serum creatinine levels (166.19 μmol / L), indicating a direct correlation between above-therapeutic concentrations (>2.3 μg / mL) of polymyxin E and markers of renal impairment. This further emphasizes the crucial role of therapeutic drug monitoring (TDM) in preventing dose-related toxicities and maintaining effectiveness against multidrug-resistant pathogens. This method exhibits 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 protocol for clinical TDM.

[0179] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present disclosure and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present 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 includes: pretreating a plasma sample with HLB magnetic beads to obtain a sample to be tested; then using liquid chromatography-tandem mass spectrometry to quantify the content of polymyxin E in the sample to be tested; before pretreating the plasma sample with the HLB magnetic beads, the HLB magnetic beads are activated with methanol; the polymyxin E is polymyxin E1 and polymyxin E2. The steps for pre-treating plasma samples using HLB magnetic beads to obtain the test sample include: S1': Plasma sample pretreatment; Take a plasma sample, add internal standard working solution and diluent, vortex mix to obtain plasma sample pretreatment solution; 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, equilibration, adsorption, rinsing and elution steps are performed sequentially by the magnetic bead extractor, and the eluent is collected as the sample to be tested; The automated extraction of plasma sample pretreatment solution involves preloading activated magnetic beads into wells 1 and 7 of the magnetic bead extraction plate, preloading ultrapure water into wells 2, 8, 4, and 10 for magnetic bead equilibration and rinsing, preloading the plasma sample pretreatment solution obtained in step S1' into wells 3 and 9, preloading methanol into wells 5 and 11 for magnetic bead cleaning, and preloading eluent into wells 6 and 12. The magnetic bead extraction instrument sequentially performs the steps of magnetic bead activation, magnetic bead equilibration, sample loading and adsorption, rinsing, washing, and elution. Finally, the eluent from wells 6 and 12 is collected, and the supernatant is obtained by centrifugation to obtain the sample to be tested. The eluent mentioned in step S2' is a 5% formic acid-30% acetonitrile aqueous solution.

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

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

4.

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

5. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry as described in claim 4, characterized in that, The quantitative ion pairs of polymyxin E1 are 390.900 and 385.000; the qualitative ion pairs of polymyxin E1 are 390.900 and 101.100; the quantitative ion pairs of polymyxin E2 are 386.100 and 380.100; the qualitative ion pairs of polymyxin E2 are 386.100 and 101.100; and the quantitative ion pairs of polymyxin B1 are 602.569 and 241.

100.

6. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry as described in claim 1, characterized in that, The method for activating the HLB magnetic beads with methanol is as follows: take HLB magnetic beads, add methanol, the mass-to-volume ratio of HLB magnetic beads to methanol is 5 mg / ml, vortex mix, and obtain the activated magnetic bead solution.

7. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry as described in claim 1, characterized in that, Includes the following steps: S1: Prepare a series of standard working solutions, quality control solutions, and internal standard working solutions; the series of standard working solutions are prepared using blank plasma to prepare a series of polymyxin E1 and polymyxin E2 mixed standard working solutions with a concentration range of 0.01~20μg / mL; the series of quality control solutions are prepared 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 0.2~8μg / mL; S2: Pretreatment standard working solution and quality control solution: Take the standard working solution or quality control solution obtained in step S1, add the internal standard working solution and diluent, vortex mix well to obtain the pretreatment standard working solution or pretreatment quality control solution. S3: Automated extraction of the pretreatment standard working solution and pretreatment quality control solution obtained in step S2 and the plasma sample pretreatment solution obtained in step S1' to obtain the standard working solution to be tested, the quality control solution to be tested, and the sample to be tested; S4: Inject the working solution of the standard to be tested prepared in step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Use the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio as the Y-axis to perform linear regression analysis to obtain the standard curve and the standard curve equation. S5: Inject the sample to be tested obtained in step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the standard curve and standard curve equation obtained in step S4, quantify polymyxin E1 and polymyxin E2 in the sample to be tested. S6: Inject the test quality control solution from step S3 into liquid chromatography-tandem mass spectrometry for detection and analysis. Based on the standard curve and standard curve equation obtained in step S4, calculate the amounts of polymyxin E1 and polymyxin E2 in the test quality control solution. Compare the accuracy and precision with the theoretical contents of polymyxin E1 and polymyxin E2 in the test quality control solution. If the deviation of accuracy and precision is within the predetermined acceptance standard, the detection result of step S5 is valid. If the deviation of accuracy and precision exceeds the predetermined acceptance standard, re-detect according to steps S1 to S6.

8. The method for detecting polymyxin E in plasma based on magnetic bead extraction combined with liquid chromatography-tandem mass spectrometry as described in claim 7, characterized in that, The predetermined acceptance criteria mentioned in step S6 are a deviation within ±15% and an RSD of less than 15%.

Images