Method for detecting plasma concentration of polymyxin E

Abstract

The invention discloses a method for detecting the plasma concentration of polymyxin E. L-PME1 is used as an internal standard, a liquid chromatography-tandem mass spectrometry detection method is adopted for detection, the extraction efficiency and the mass spectrometry ionization efficiency are far superior to those of a common structural analogue internal standard, and a high internal standard synchronization rate can be kept in different matrixes. In addition, the L-PME1 does not exist in a natural polymyxa fermentation product, so that cross influence cannot be generated.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a method for detecting the blood drug concentration of polymyxin E. Background Art

[0002] Polymyxins are a collective term for a series of peptide antibiotics isolated from the culture of Bacillus polymyxa strains. They were first recognized as having antibacterial activity in the 1940s. Although five different structurally polymyxins (polymyxin A, B, C, D, E) were initially isolated, due to reasons such as antibacterial activity and toxicity, only polymyxin B (PMB) and polymyxin E (PME) were finally applied clinically.

[0003] Chemically, PME is a cyclic peptide composed of 10 amino acids (such as Figure 1 ). Among them, the 6th amino acid is D - type leucine, and the rest are L - type amino acids. The drug forms of PME mainly include polymyxin E and polymyxin E methanesulfonate. Among them, polymyxin E methanesulfonate binds methanesulfonic acid to the free amino groups of the 1st, 3rd, 5th, 8th, and 9th diaminobutyric acids, and has relatively low antibacterial activity. It needs to be metabolized into polymyxin E in the body to exert antibacterial activity. According to the different fatty acid structural formulas connected to the 1st amino acid, there are various isomers such as PME1, PME2, PME3, PME4, etc. Some PME are prone to changes in the leucine at the 7th position, forming rare isomers such as PME1 - ileu, PME1 - val, PME1 - NVa, PME2 - ile, PME2 - val, PME8 - ile. The main components in commercially available PME injection are PME1 and PME2.

[0004] PME began to be applied clinically in the 1950s, but was abandoned in the 1970s due to high renal toxicity, neurotoxicity, and narrow antibacterial spectrum. With the abuse of antibiotics, there are more and more drug - resistant bacteria, and polymyxins have played an important role in the clinical treatment of extensively drug - resistant Gram - negative bacilli, especially multi - drug - resistant (MDR) and extensively drug - resistant (XDR) Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae infections. Since PME is a highly nephrotoxic drug, and acute kidney injury (AKI) often occurs at conventional doses. Given that both the effectiveness and safety (renal toxicity, neurotoxicity, etc.) of PME are closely related to the dose and blood drug concentration, and it has the characteristic of a narrow therapeutic window (low therapeutic index), it is very necessary to guide the clinical application of PME through therapeutic drug monitoring (TDM) to avoid adverse reactions and improve clinical efficacy.

[0005] Currently, the common methods for therapeutic drug concentration monitoring are mainly liquid chromatography and immunological methods. However, due to reasons such as sensitivity, specificity, and cross-reactivity, it is difficult to apply them to the blood drug concentration monitoring of PME. Currently, the common monitoring method in clinical practice is liquid chromatography-tandem mass spectrometry. However, since PME is easily interfered by matrix effects, it is necessary to calibrate the matrix effects through internal standards. The best internal standard for liquid chromatography-tandem mass spectrometry is the isotope-labeled analyte. The internal standard has exactly the same physicochemical properties as the analyte and can effectively calibrate the matrix effects of the analyte. However, since PME is a macromolecular substance with a molecular weight exceeding 1000, it is difficult to synthesize by chemical methods. Therefore, there is no isotope-labeled polymyxin internal standard worldwide.

[0006] Some studies selected structurally similar cyclic antibiotics as internal standards, or even PMB as an internal standard. However, due to the differences in physicochemical properties, the synchronization rate of internal standard calibration is poor and cannot meet the needs of clinical monitoring. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting the blood drug concentration of polymyxin E. Using L-PME1 as an internal standard, it is detected by liquid chromatography-tandem mass spectrometry, which is far superior to common structurally similar internal standards in terms of extraction efficiency and mass spectrometry ionization efficiency, and can also maintain a high internal standard synchronization rate in different matrices. In addition, L-PME1 does not exist in natural polymyxin fermentation products, so there will be no cross-interference.

[0008] The technical solution adopted by the present invention to solve its technical problems is: A method for detecting the blood drug concentration of polymyxin E, comprising the following steps: (1) Sample pretreatment: Take 100 μL of serum sample, add 10 μL of internal standard working solution, mix evenly, then add 300 μL of sample release agent, and perform cryo-centrifugation at 15000 g for 10 min. Take the supernatant for testing; the internal standard working solution is a 10 μg / mL aqueous solution of L-PME1; (2) Liquid chromatography-tandem mass spectrometry detection: The supernatant is introduced into a liquid chromatography-tandem mass spectrometer for detection, thereby detecting the blood drug concentration of polymyxin E.

[0009] The structure of PME1 consists of 10 amino acids, including 6 rare amino acids, 2,4-diaminobutyric acid (Dab). The amino acid at position 10 of PME1 binds to the Dab at position 4 to form a ring. Among the above amino acids, except for the leucine at position 6 which is a D-type amino acid, the others are all L-type amino acids (Development of new polymyxin derivatives for multi-drugresistant Gram-negative infections, The Journal of Antibiotics (2017) 70, 386–394).

[0010] The DL isomers of amino acids usually have optical activity, resulting in the optical rotation of polarized light. Generally speaking, in liquid chromatography, especially in conventional reversed-phase chromatography, it is impossible to chromatographically separate DL isomers.

[0011] In the present invention, after swapping the D-type and L-type amino acids at positions 6, 7, and 10, it will cause differences in the polarity of the molecule, resulting in changes in the retention time in reversed-phase chromatography. The possible reason is that the side chains of the above amino acids are wrapped inside the seven-membered polypeptide ring or exposed outside in the DL isomers, changing the polarity of the molecular surface. Among them, the polarity changes caused by the DL changes of the amino acids at positions 7 and 10 are relatively small. Although there are differences in retention time, they are not sufficient to produce baseline separation. However, the inventor unexpectedly found that after changing the D-type leucine at position 6 to L-type leucine, the difference in chromatographic retention time is relatively large, and the two can produce baseline separation. Therefore, although D-PME1 (referring to PME1 with D-type leucine at position 6) and L-PME1 are isomers, they can be distinguished by chromatographic separation and meet the basic conditions for becoming internal standards.

[0012] As isomers, D-PME1 and L-PME1 have highly similar physical and chemical properties, especially in terms of extraction efficiency and mass spectrometry ionization efficiency, which are far superior to common structural analog internal standards, and can also maintain a high internal standard synchronization rate in different matrices. In addition, L-PME1 does not exist in natural polymyxin fermentation products, so there will be no cross-interference.

[0013] The sample release agent is obtained by mixing 0.1 mL of formic acid into 100 mL of methanol.

[0014] The chromatographic conditions for liquid chromatography-tandem mass spectrometry detection are as follows: Chromatographic column: C18 polypeptide column, 2.1×100 mm, pore size Mobile phase A: ultrapure water containing 0.1% formic acid; Mobile phase B: acetonitrile; gradient elution; Flow rate: 0.4 mL / min; Injection volume: 1 - 5 μL.

[0015] The gradient elution parameters are set as follows: At the start, the proportion of mobile phase B is 15%; At 5 min, the proportion of mobile phase B is 25%; At 5.1 min, the proportion of mobile phase B is 100%; At 5.6 min, the proportion of mobile phase B is 100%; At 5.7 min, the proportion of mobile phase B is 15%; At 7.7 min, the proportion of mobile phase B is 15%.

[0016] The mass spectrometry conditions for liquid chromatography - tandem mass spectrometry detection are as follows: For the analyte PME1: parent ion 585.1, daughter ions 241.1; 101.1; For the analyte PME2: parent ion 578.6, daughter ions 227.1; 101.1; For the analyte L - PME1: parent ion 585.1, daughter ion 101.1.

[0017] The beneficial effects of the present invention are: in terms of extraction efficiency and mass spectrometry ionization efficiency, it is far superior to common structurally similar internal standards, and can maintain a high internal standard synchronization rate in different matrices. In addition, L - PME1 does not exist in natural polymyxin fermentation products, so there will be no cross - influence. Description of the Drawings

[0018] Figure 1 It is a chemical structure pattern diagram of PME; Figure 2 It is a chromatogram of L - PME1 and common clinically used polymyxin reference substances; Figure 3 It is a chromatogram for the study of polymyxin internal standard synchronization rate; Figure 4 It is the test result of the internal standard synchronization rate. Detailed Embodiments

[0019] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0020] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0021] Example 1: 1. Preparation of reagents Sample release agent: Add 0.1 ml of formic acid to 100 ml of methanol and mix evenly; Internal standard working solution: Pipette an appropriate amount of L-PME1 (Sangon Biotech (Shanghai) Co., Ltd.) stock solution and dilute it with water to 10 μg / ml.

[0022] 2. Sample pretreatment method Take 100 μl of serum sample, add 10 μl of internal standard working solution, mix well, then add 300 μl of sample releasing agent, centrifuge at 15000 g for 10 min under freezing conditions, and take the supernatant for testing.

[0023] 3. Standard curve Take an appropriate amount of PME1 (Sangon Biotech (Shanghai) Co., Ltd.) and dilute it with water to a standard curve solution of 2 - 200 μg / ml. Subsequently, take 10 μl of the standard curve solution, add it to 90 μl of blank serum, add 10 μl of internal standard working solution, mix well, then add 300 μl of sample releasing agent, centrifuge at 15000 g for 10 min under freezing conditions, and take the supernatant for testing.

[0024] 4. Liquid chromatography - tandem mass spectrometry detection Liquid chromatography - tandem mass spectrometer: LCMS - 8040CL (Shimadzu, Japan) Chromatographic column: C18 special column for polypeptides (ACQUITY UPLC BEH C18 column), 2.1 x 100 mm, pore size Mobile phase A: Ultra - pure water containing 0.1% formic acid Mobile phase B: Acetonitrile Flow rate: 0.4 ml / min Injection volume: 1 - 5 μl (adjusted according to the instrument sensitivity, 2 μl is selected in this example).

[0025] Mass spectrometry parameters: Ionization mode: ESI+ Ion source temperature: 150 °C Capillary voltage: 3.5 kV Desolvation gas flow rate: 750 L / hr Desolvation temperature: 500 °C Cone - hole counter - blowing gas flow rate: 150 L / hr.

[0026] The elution gradient is shown in Table 1, and the mass spectrometry MRM parameters are shown in detail in Table 2. Other parameters are the default parameters of the instrument.

[0027] Table 1: Liquid chromatography elution gradient Time Proportion of Mobile Phase B Start 15％ 5 min 25％ 5.1 min 100％ 5.6 min 100％ 5.7 min 15％ 7.7 min 15％ 。

[0028] Table 2: Mass spectrometry MRM parameters Analyte Parent Ion Daughter Ion PME1 585.1 241.1；101.1* PME2 578.6 227.1；101.1* L-PME1 585.1 101.1* Note: The channels marked with * are quantitative ion channels.

[0029] Example 2: Chromatographic separation of L-PME1 and the analyte To confirm that L-PME1 does not interfere with the analyte, a mixed solution of PMB1, PMB2, PME1, PME2, and L-PME1 with a concentration of 1 μg / ml was prepared in this study and detected using the method of Example 1. PMB1, PMB2, PME1, and PME2 are all from Sangon Biotech (Shanghai) Co., Ltd.

[0030] The detection results are as Figure 2 shown. There are significant differences in the retention times of L-PME1 and PME1, and baseline separation can be achieved. Even though the above two are isomers and have the same MRM channels, they can be separated by chromatography without mutual interference.

[0031] It is worth mentioning that the retention times of L-PME1 and polymyxin B2 (PMB2) are the same. However, there are significant differences in their molecular weights, and they can be separated using different MRM channels without mutual interference.

[0032] In summary, L-PME1 does not interfere with the analyte and meets the requirements for the selection of internal standards in clinical detection.

[0033] Example 3: Comparison of the synchronization rates of internal standards in different matrices Although the types of clinical specimens are relatively single, all being serum samples, due to various pathological states of patients, the matrix will change greatly. Especially, polymyxin drugs have strong nephrotoxicity, which can cause significant changes in some renal function index substances (such as creatinine). These substances will significantly affect the detection of PME in two aspects: 1. PME has a high protein binding rate. Since PME itself is a cyclic decapeptide, its physicochemical properties are somewhat similar to those of binding proteins, so it is prone to co-precipitation during the precipitation of sample proteins.

[0034] 2. PME is prone to matrix interference during the mass spectrometry ionization process. There are many impurities in the purified sample, which will co-elute with the analyte and internal standard during chromatographic separation and interact with each other during the ionization of the mass spectrometry ion source, including ion gain or ion suppression.

[0035] These matrix effects will affect both the analyte PME and the internal standard. When the degree of influence of the internal standard by the matrix effect is inconsistent with that of PME, it will not only not correct the matrix effect but also further increase the error.

[0036] Therefore, in this study, 20 negative sera of patients who had not taken drugs were randomly selected, and the analytes PME1 and PME2, the internal standard L-PME1 selected in the present invention, and the commonly used internal standards in the literature, daptomycin, vancomycin, norvancomycin, vancomycin impurity C, teicoplanin, and polymyxin B1 were added thereto for comparison. The total ion current chromatograms are as follows Figure 3 as shown. The MRM parameters of all analytes and candidate internal standards are shown in Table 3

[0037] Table 3: MRM parameters of analytes and candidate internal standards Chinese English Parent Ion Daughter Ion Vancomycin Vancomycin 725.5 144.1、100.0 Nor-Vancomycin Nor-Vancomycin 718.5 144.1、100.2 Teicoplanin Teicoplanin 940.6 316.2、144.1 Polymyxin B1 Polymyxin B1 602.6 241.1、101.0 L-Polymyxin E1 Polymyxin E1 585.6 241.1、101.1 Polymyxin E1 Polymyxin E1 585.6 241.1、101.1 Polymyxin E2 Polymyxin E2 578.6 227.1、101.1 Daptomycin Daptomycin 810.9 159.1、341.2 .

[0038] The spiked concentrations of the above-mentioned analytes and candidate internal standards were all 1 μg / ml. 100 μl of the spiked sample was added to 300 μl of the sample releasing agent, and centrifuged at a centrifugal force of 15000 g for 10 min at low temperature. The supernatant was taken for detection

[0039] In this study, aqueous solutions of 1 μg / ml analytes and candidate internal standards were prepared as reference substances. 100 μl of the reference substance was mixed with 300 μl of the sample releasing agent and then injected for detection. Taking the peak area of the reference substance as 100%, the absolute recovery rate of each analyte in each sample was calculated. The ratio of the absolute recovery rate of the analyte to the internal standard was the internal standard synchronization rate

[0040] The results are as Figure 4 shown. The internal standard synchronization rates of vancomycin, norvancomycin, and daptomycin were significantly lower than the normal values. The possible reason is that PME is easily bound by albumin in the serum and co-precipitated by the sample releasing agent, while the co-precipitation phenomenon of the above three internal standards is lower. The synchronization rate of teicoplanin was close to 80%, but the standard deviation of its synchronization rate in 20 matrices was large, so it was not suitable as an internal standard either

[0041] The internal standard synchronization rates of PMB1 and L-PME1 were both between 90-100%, and the standard deviation was <5%, so they were suitable as internal standards. Among them, the internal standard synchronization rate of L-PME1 was significantly higher than that of PMB1, and the difference between the two was about 5%. The synchronization rates of L-PME1 with PME1 and PME2 were as high as 100.3% and 99.9%, respectively, which were the best internal standards for the above two analytes

[0042] Example 4: Methodological verification - linearity To prove the effectiveness of the present invention, in this study, the standard curve described in Example 1 was processed and then detected. A standard curve was established with the peak area ratio of the analytes (PME1 and PME2) to the internal standard (L-PME1) as the abscissa and the concentration as the ordinate. The results are shown in Table 4. Both analytes showed good linearity in the low and high linear ranges, and the correlation coefficient r2 was higher than 0.99, meeting the requirements of clinical detection, which proved that L-PME1 was suitable as an internal standard.

[0043] Table 4: Linearity of PME1 and PME2

[0044] Example 5: Methodology Verification - Recovery In this study, serum spiked samples with concentrations of 0.3, 3, and 8 μg / ml were prepared, processed according to the method described in Example 1, and then detected. The standard curve established in Example 5 was used to calculate the sample recovery rate. Each sample was detected in parallel 7 times, and the average recovery rate and coefficient of variation were calculated.

[0045] The results are shown in Table 5. Satisfactory detection results could be obtained for both analytes at the three spiked concentrations. The recovery rate was between 90% and 110%, and the coefficient of variation was <10%, meeting the requirements of clinical detection, which proved that L-PME1 was suitable as an internal standard.

[0046] Example 6: Methodology Verification - Precision In this study, the same spiked samples as in Example 4 and Example 5 were used. Three experimenters independently established the standard curve and detected the samples. Each sample was detected 7 times, and the coefficient of variation among the 21 samples was statistically analyzed to evaluate the method precision.

[0047] The results are shown in Table 6. The coefficient of variation among the 21 samples from 3 independent experimenters was <10%, which proved that the method had good precision, met the clinical requirements, and also confirmed that L-PME1 was suitable as an internal standard.

[0048] The above-described embodiments are only a preferred solution of the present invention, and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A method for detecting the blood drug concentration of polymyxin E, characterized in that, It includes the following steps: (1) Sample pretreatment: Take 100 μL of serum sample, add 10 μL of internal standard working solution, mix well, then add 300 μL of sample releasing agent, centrifuge at 15000 g of centrifugal force for 10 min under freezing conditions, and take the supernatant for testing; the internal standard working solution is an aqueous solution of L-PME1 at 10 μg / mL. (2) Liquid chromatography-tandem mass spectrometry detection: The supernatant is introduced into a liquid chromatography-tandem mass spectrometer for detection, so as to detect the blood drug concentration of polymyxin E.

2. The method according to claim 1, characterized in that, The sample releasing agent is obtained by mixing 0.1 mL of formic acid into 100 mL of methanol.

3. The method according to claim 1, wherein The chromatographic conditions for liquid chromatography-tandem mass spectrometry detection are as follows: Chromatographic column: C18 polypeptide column, 2.1×100 mm, pore size Mobile phase A: ultrapure water containing 0.1% formic acid; Mobile phase B: acetonitrile; gradient elution; Flow rate: 0.4 mL / min; Injection volume: 1 - 5 μL.

4. The method according to claim 3, wherein The gradient elution parameter settings are as follows: At the beginning, the proportion of mobile phase B is 15%; At 5 min, the proportion of mobile phase B is 25%; At 5.1 min, the proportion of mobile phase B is 100%; At 5.6 min, the proportion of mobile phase B is 100%; At 5.7 min, the proportion of mobile phase B is 15%; At 7.7 min, the proportion of mobile phase B is 15%.

5. The method according to claim 1, characterized in that, The mass spectrometry conditions for liquid chromatography-tandem mass spectrometry detection are as follows: Analyte PME1: parent ion 585.1, daughter ions 241.1; 101.1; Analyte PME2: parent ion 578.6, daughter ions 227.1; 101.1; Analyte L-PME1: parent ion 585.1, daughter ion 101.1.

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