A method for simultaneous detection of polymyxin b and polymyxin e

Abstract

This invention relates to a method for the simultaneous detection of polymyxin B and polymyxin E, comprising the following steps: (1) using isotopically labeled polymyxins B1, B2, E1, and E2 as internal standards, dissolving them, and diluting them with methanol containing formic acid to prepare a sample release agent; (2) mixing the test sample with the above sample release agent, vortexing, allowing it to stand, centrifuging, and then injecting the supernatant into the sample for detection using liquid chromatography-tandem mass spectrometry. This invention enables the simultaneous detection of patient samples containing both polymyxin B and E.

Description

Technical Field

[0001] This invention belongs to the field of polymyxin detection technology, and specifically relates to a method for simultaneously detecting polymyxin B and polymyxin E. Background Technology

[0002] Polymyxin B and polymyxin E, two classes of polypeptide antibiotics extracted from the fermentation broth of *Paenibacillus polymyxa*, are renowned for their exceptional antibacterial efficacy. They have been widely used in clinical treatment since the 1950s, although they were gradually replaced by other, safer antibiotics due to their significant nephrotoxicity. However, against the backdrop of increasingly severe infections caused by multidrug-resistant and extensively drug-resistant Gram-negative bacilli, polymyxins, with their unique antibacterial activity, have once again become a powerful weapon in clinical treatment.

[0003] Polymyxin B and polymyxin E have similar antibacterial spectra, both belonging to the narrow-spectrum antibiotics. They show significant antibacterial effects against most Gram-negative bacilli, especially against drug-resistant strains such as Pseudomonas aeruginosa and Acinetobacter spp. Therefore, they play a crucial role in treating serious infections caused by drug-resistant Gram-negative bacteria, such as bacteremia, endocarditis, pneumonia, and post-burn infections. In addition, polymyxin B and polymyxin E are also widely used to treat various conditions including urinary tract infections, eye infections, tracheal infections, meningitis, sepsis, burn infections, and skin and mucous membrane infections. Given their potential nephrotoxicity, the clinical use of polymyxins must strictly adhere to indications, and drug concentration monitoring should be performed when necessary to ensure the safety and effectiveness of treatment.

[0004] Chemically, polymyxin B and polymyxin E are polypeptides composed of multiple amino acids, exhibiting remarkable structural similarity, differing only in the sixth amino acid position of the peptide ring: polymyxin B contains phenylalanine, while polymyxin E contains leucine. It is noteworthy that polymyxins are not single compounds but complex mixtures produced through fermentation, containing multiple components. For example, polymyxin B contains components such as B1, B2, B3, B1-I, B4, and B5 in varying proportions. Among these components, B1 and B2 are present in the highest amounts in polymyxin B and contribute the most to its efficacy and toxicological effects. Polymyxin E exhibits a similar pattern. Therefore, therapeutic drug monitoring (TDM) primarily focuses on the concentrations of the four polymyxin components: B1, B2, E1, and E2.

[0005] In clinical practice, serum or plasma samples are typically collected for therapeutic drug monitoring (TDM) of polymyxins. Currently, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is the most widely used detection technique. This technique generally involves treatment with organic solvents to precipitate proteins and release polymyxins, followed by separation of the target analyte from impurities using a C18 column. On a triple quadrupole mass spectrometer, multiple reaction monitoring (MRM) mode is used for selective detection of specific ion pairs of polymyxins. This method exhibits high specificity, accurately distinguishing the various components of polymyxins; simultaneously, it provides accurate and precise quantitative results over a relatively wide linear range, meeting the requirements of clinical therapeutic drug monitoring.

[0006] In liquid chromatography-tandem mass spectrometry (LC-MS / MS), internal standards are an indispensable key factor. Internal standards play a dual role in sample analysis: firstly, they calibrate for losses during sample pretreatment; secondly, they compensate for fluctuations in ionization efficiency. Therefore, an ideal internal standard should be a substance that is highly similar in physicochemical properties to the target analyte and is absent in the sample. The most ideal internal standard is an isotope-labeled compound of the target analyte, which not only possesses identical physicochemical properties to the target analyte but also does not naturally exist in the sample and can be effectively distinguished by mass spectrometry.

[0007] Because isotopically labeled internal standards for polymyxin B and polymyxin E are difficult to obtain, a strategy of using each other as internal standards is often employed in research. That is, when determining polymyxin B, polymyxin E is used as the internal standard, and vice versa. Due to the unique physicochemical properties of polymyxins, no other substitutes with similar physicochemical properties have yet been found.

[0008] In traditional diagnostic procedures, polymyxin B and polymyxin E are usually used alone, so using a mutual internal standard strategy does not cause interference. However, with the evolution of clinical treatment strategies, the combined intravenous and topical administration of polymyxin B and polymyxin E has gradually become the norm. In such combined use, the mutual internal standard strategy is no longer applicable. Furthermore, although polymyxin B and polymyxin E are highly similar in structure, subtle differences still exist between them. These differences may lead to variations in physicochemical properties, thereby affecting the stability of test results, especially in certain specific samples.

[0009] Given that previous methods employed non-isotope-labeled internal standard strategies, in-depth research into the absolute recovery rate of polymyxins is crucial to ensure it is as close to 100% as possible. This necessitates precise and complex sample pretreatment steps, typically including acidification and precipitation. This undoubtedly increases the operational complexity of clinical testing and may introduce additional uncertainties in the test results. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting polymyxin B and polymyxin E, which can simultaneously detect patient samples containing both polymyxin B and E.

[0011] This invention provides a method for simultaneously detecting polymyxin B and polymyxin E, comprising the following steps:

[0012] (1) Isotope-labeled polymyxins B1, B2, E1, and E2 were used as internal standards. After dissolution, they were diluted with methanol containing formic acid to prepare sample release agents.

[0013] (2) Mix the test sample with the above sample release agent, vortex, let stand, centrifuge, take the supernatant and inject it into the sample, and use liquid chromatography-tandem mass spectrometry for detection.

[0014] Preferably, in step (1), the isotopically labeled polymyxins B1, B2, E1, and E2 are derived by using leucine at position 7 of natural polymyxin. 13 C6 15 N-labeled isotopic leucine substitution.

[0015] Preferably, the formic acid content in the methanol in step (1) is 0.1-0.5 v / v%.

[0016] Preferably, the volume ratio of the test sample to the sample release agent in step (2) is 1:3-5.

[0017] Preferably, the parameters of the liquid chromatography-tandem mass spectrometry in step (2) are as follows: mobile phase A: ultrapure water containing 0.1 v / v% formic acid; mobile phase B: acetonitrile; flow rate: 0.4 ml / min; column temperature: 40 °C.

[0018] Beneficial effects

[0019] (1) The present invention uses isotopically labeled polymyxins B1, B2, E1, and E2 as internal standards. Their physicochemical properties are exactly the same as those of naturally occurring polymyxins B1, B2, E1, and E2, which can effectively correct the variation of samples in the pretreatment and ionization stages, thereby ensuring the accuracy and precision of the detection results.

[0020] (2) The present invention can simultaneously detect patient samples containing both polymyxin B and E.

[0021] (3) Given the adoption of the isotope-labeled internal standard strategy, the present invention simplifies the sample pretreatment process, and only one step is required to add the sample, thereby improving the efficiency and convenience of detection. Attached Figure Description

[0022] Figure 1 This is the calibration curve for polymyxin B1.

[0023] Figure 2 This is the calibration curve for polymyxin B2.

[0024] Figure 3 This is the calibration curve for polymyxin E1.

[0025] Figure 4 This is the calibration curve for polymyxin E2.

[0026] Figure 5 The absolute recovery rate of polymyxin in different extracts is given. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] Example 1

[0029] I. Preparation of Sample Release Agent

[0030] The isotopically labeled polymyxins B1, B2, E1, and E2 were used as internal standards (the isotopically labeled polymyxins B1, B2, E1, and E2 are derived from the leucine residue at position 7 of the natural polymyxin). 13 C6 15 The N-labeled isotope leucine was dissolved in water to prepare stock solutions with a concentration of 1 mg / mL. Appropriate amounts of each polymyxin stock solution were mixed with methanol containing 0.1 v / v% formic acid to achieve an internal standard concentration of 333 ng / mL for each polymyxin.

[0031] II. Sample Preprocessing

[0032] ① Take 50 μl of sample and add 150 μl of sample release agent containing internal standard;

[0033] ② Vortex for 5 seconds, let stand for 5 minutes, then vortex again for 5 seconds;

[0034] ③ Centrifuge for 5 minutes in a centrifuge with a centrifugal force of over 12000×g and a temperature below 10℃;

[0035] ④ Collect the supernatant for subsequent testing.

[0036] III. Instrument Conditions

[0037] Mobile phase A: Ultrapure water containing 0.1% formic acid;

[0038] Mobile phase B: Acetonitrile;

[0039] Mobile phase ratio: Elution gradient details are shown in the table below.

[0040] time mobile phase B percentage Start 5 1min 100 1.4min 100 1.5min 5 3.5min 5

[0041] Flow rate: 0.4 ml / min;

[0042] Column temperature: 40℃.

[0043] Because polymyxin exhibits a multi-charge distribution in mass spectrometry, the highest-response precursor ion varies across different instruments. Therefore, it is necessary to compare response values ​​and eliminate cross-interference to determine the ion channel. The following are the precursor and daughter ion parameters for three common ionization modes.

[0044] [M+4H] Mass Spectrometry Multiple Reaction Monitoring Parameters

[0045]

[0046] [M+3H] Mass Spectrometry Multiple Reaction Monitoring Parameters

[0047]

[0048]

[0049] [M+2H] Mass Spectrometry Multiple Reaction Monitoring Parameters

[0050]

[0051] IV. Linear Range

[0052] In this study, polymyxin B and E standard substances were prepared into stock solutions and then added to serum at different ratios to achieve concentrations of 0.12, 0.6, 3, 6, 12, and 24 μg / mL. The concentrations of polymyxin B1, B2, E1, and E2 were calculated according to the instructions of the standard substances.

[0053] The spiked samples were pretreated according to the method described in this invention before being injected for detection. A calibration curve was established with the peak area ratio of the standard substance to the internal standard as the ordinate and the concentration as the abscissa. The results are as follows: Figures 1-4 The results showed that the method exhibited good linearity in the range of 0.12–24 μg / mL (correlation coefficient R0). 2 >0.99).

[0054] V. Recovery Rate

[0055] In this study, three spiked samples of polymyxin B and E at different concentrations were prepared for detection, and the samples were divided into three batches, with seven replicates for each test. The recoveries are shown in the table below, all between 100% and 105%.

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[0065]

[0066] VI. Precision

[0067] In this study, three spiked samples of polymyxin B and E at different concentrations were prepared for testing. The samples were divided into three batches, with seven replicates per batch. The spiked concentrations and measured results are shown in the table below. The calculated coefficients of variation for all results were less than 8.90%.

[0068]

[0069]

[0070]

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[0074]

[0075]

[0076] Example 2

[0077] Comparison of recovery rates of different extraction reagents

[0078] This study prepared spiked samples of polymyxins B and E and used different extraction reagents for pretreatment. The applicability of methanol and acetonitrile as protein precipitants was compared, and the applicability of formic acid, trichloroacetic acid (TCA), and ammonium acetate as additives was also verified. The absolute recovery rate of polymyxins was calculated by adding an internal standard to the supernatant after sample extraction and separation; this was used only to correct for ionization efficiency and not for extraction loss.

[0079] The results are as follows Figure 5 The results showed that methanol containing 0.1% formic acid (i.e., the extract formulation of this invention) had the highest recovery rate, approaching 100% absolute recovery. Other extraction reagents all showed varying degrees of recovery loss.

[0080] Example 3

[0081] Comparison of matrix effects using isotope internal standards and mutual internal standards strategies

[0082] This study collected blank plasma samples from six different individuals. After treatment with a sample release agent without internal standards, the supernatant was used to add polymyxin B, polymyxin E, and isotopically labeled polymyxins B1 and B2 for mass spectrometry analysis. Two strategies were employed to calculate the matrix factor and normalized matrix factor: using the isotope as the internal standard for polymyxin B and using polymyxin E as the internal standard for polymyxin B.

[0083] The results showed that matrix 3 caused an abnormally high response value of polymyxin B (matrix gain). Isotope-labeled polymyxins B1 and B2 could effectively correct for matrix gain. When polymyxin E was used as an internal standard, the asynchrony of matrix gain amplitudes led to abnormal detection results.

[0084]

[0085]

Claims

1. A method for simultaneously detecting polymyxin B and polymyxin E, comprising the following steps: (1) Isotope-labeled polymyxins B1, B2, E1, and E2 were used as internal standards. After dissolution, they were diluted with methanol containing formic acid to prepare sample release agents. (2) Mix the test sample with the above sample release agent, vortex, let stand, centrifuge, take the supernatant and inject it into the sample, and use liquid chromatography-tandem mass spectrometry for detection.

2. The method according to claim 1, characterized in that: The isotopically labeled polymyxins B1, B2, E1, and E2 in step (1) are derived by using leucine at position 7 of natural polymyxin. 13 C6 15 N-labeled isotopic leucine substitution.

3. The method according to claim 1, characterized in that: The formic acid content in the methanol in step (1) is 0.1-0.5 v / v.

4. The method according to claim 1, characterized in that: The volume ratio of the test sample to the sample release agent in step (2) is 1:3-5.

5. The method according to claim 1, characterized in that: The parameters for liquid chromatography-tandem mass spectrometry in step (2) are as follows: mobile phase A: ultrapure water containing 0.1 v / v% formic acid; mobile phase B: acetonitrile; flow rate: 0.4 ml / min; column temperature: 40 ℃.

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