A method for detecting polymyxin E1 and E2 in plasma by LC-MS / MS

Abstract

The present invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for detecting polymyxin E1 and E2 in plasma using LC-MS / MS. The method provided by the present invention has simple pretreatment steps, requires a small amount of plasma, can complete the detection of multiple samples in a relatively short time, has high pretreatment efficiency, uses simple reagent preparation, has low pretreatment costs, requires relatively low testing personnel, is highly operational, and is suitable for large-scale application.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting polymyxin E1 and E2 in plasma using an LC-MS / MS method. Background Art

[0002] Polymyxin E is a polypeptide antibacterial drug containing multiple different components, with a mechanism of action and antimicrobial spectrum similar to polymyxin B. The emergence of multidrug-resistant (MDR) Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, and the development of fewer new and effective antibacterial drugs, have led to the reappearance of polymyxin E in clinical practice, primarily for intestinal infections caused by Escherichia coli, Salmonella, Shigella, Escherichia coli, and Brucella.

[0003] Polymyxin E is a complex compound, primarily consisting of polymyxin E1 and polymyxin E2. Polymyxin E blood concentrations are closely associated with nephrotoxicity and antibacterial efficacy. Polymyxin E pharmacokinetics vary significantly in critically ill patients, making blood concentration monitoring particularly important.

[0004] The Chinese Journal of Infection and Chemotherapy published a method for determining the concentrations of polymyxin E1 and E2 in human plasma using liquid chromatography-tandem mass spectrometry. This method used solid-phase extraction for plasma pretreatment, with a mobile phase of 0.1% formic acid solution: acetonitrile-methanol, and an analysis time of 3.5 minutes. However, this method had a low recovery rate of only 29.2%-33.0%. The low extraction efficiency and complexity of solid-phase extraction made it difficult to meet the clinical needs of collecting and testing large numbers of samples. The Chinese Journal of Clinical Pharmacology published an LC-MS / MS method for the simultaneous determination of polymyxin E and levofloxacin concentrations, also using solid-phase extraction for pretreatment, which is not suitable for testing large numbers of biological samples.

[0005] "Health Big Vision" disclosed the establishment of an LC-MS / MS method for determining the concentration of polymyxin E in human plasma. Human plasma has low short-term stability and can only ensure that the sample is stable for 18 hours when placed under wet ice conditions. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention has established a LC-MS / MS method for the rapid and accurate determination of polymyxin E1 and E2 in plasma, which meets the clinical requirements for the therapeutic drug and pharmacokinetic research of polymyxin E.

[0007] Specifically, the technical solutions of the present invention are as follows:

[0008] The present invention provides a method for rapidly and accurately determining polymyxin E1 and E2 in plasma using LC-MS / MS:

[0009] Chromatographic conditions:

[0010] The chromatographic column was ThermoAccucore aQ; mobile phase A was methanol + chloride salt; mobile phase B was water (containing 0.5% formic acid);

[0011] Gradient elution:

[0012] 0.0-0.5min: A5-15%, B 85-95%;

[0013] 0.5-3.0min: A95-100%, B 0-5%;

[0014] 3.0-3.5min: A95-100%, B 0-5%;

[0015] 3.5-3.6min: A5-15%, B 85-95%;

[0016] 3.6-5.0min: A40-60%, B 40-60%;

[0017] The flow rate was 0.4-0.6 mL / min; the column temperature was 45-55°C; and the injection volume was 5 μL.

[0018] Mass spectrometry conditions:

[0019] An electrospray ionization source was used in positive ion scan mode; source temperature: 380-420°C; spray voltage: 3400-3600 V; evaporation temperature: 380-420°C; ion transfer tube temperature: 380-420°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; and sweep gas: 10 Arbs. Scanning was performed in multiple reaction monitoring mode, and the monitoring ion transitions were m / z 585.512 → m / z 535.256 (polymyxin E1) at a collision energy of 15.99 eV, m / z 578.462 → m / z 528.489 (polymyxin E2) at a collision energy of 15.95 eV, and m / z 602.675 → m / z 100.971 (internal standard polymyxin B) at a collision energy of 36.26 eV.

[0020] Preparation of control working solution:

[0021] An appropriate amount of polymyxin E sulfate standard was accurately weighed and prepared in 20% methanol to prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 stock solutions. A series of standard working solutions were prepared using a 20% methanol solution with a gradient dilution: polymyxin E1 concentrations of 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, with low, medium, and high quality control concentrations of 1.45, 46.42, and 130.55 μg / mL, respectively. Polymyxin E2 concentrations of 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, with low, medium, and high quality control concentrations of 2.5, 80, and 225 μg / mL, respectively.

[0022] Internal standard solution:

[0023] Accurately weigh an appropriate amount of polymyxin B sulfate standard, prepare a 1000 μg / mL internal standard stock solution with 20% methanol, and then dilute it with 20% methanol to obtain a 200 μg / mL internal standard working solution.

[0024] Preparation of medicated plasma:

[0025] 5 μL of the reference working solution was transferred into 45 μL of blank plasma and vortexed for 20 s to mix. The polymyxin E1 standard curve with mass concentrations of 0.058, 0.116, 0.580, 1.160, 2.901, 5.802, 11.604, and 17.407 μg / mL was obtained. The low, medium, and high concentration quality control concentrations were 0.145, 4.642, and 13.055 μg / mL, respectively. The polymyxin E2 standard curve with mass concentrations of 0.1, 0.2, 1, 2, 5, 10, 20, and 30 μg / mL was obtained. The low, medium, and high concentration quality control concentrations were 0.25, 8.0, and 22.5 μg / mL, respectively.

[0026] Plasma sample processing:

[0027] Add 50 μL of blood sample and 5 μL of internal standard solution to the centrifuge tube in sequence, vortex for 30 seconds, add 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and adjust the pH with protein sodium salt-phosphate, vortex for 2 minutes, centrifuge at 14500 rpm for 10 minutes, take 100 μL of supernatant and add it to the injection bottle, and inject 5 μL.

[0028] In one embodiment, the LC-MS / MS method for determining polymyxin E1 and E2 in plasma is:

[0029] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0030] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.15, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0031] The chromatographic conditions of the LC-MS / MS are:

[0032] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0033] Gradient elution: 0.0-0.5 min: A 10%, B 90%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 10%, B 90%; 3.6-5.0 min: A 50%, B 50%;

[0034] The flow rate was 0.5 mL / min; the column temperature was 50°C; and the injection volume was 5 μL.

[0035] The mass spectrometry parameters of the LC-MS / MS are:

[0036] Electrospray ion source, positive ion scan mode, ion source temperature: 400°C; spray voltage: 3500 V; evaporation temperature: 400°C; ion transfer tube temperature: 400°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0037] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0038] In one embodiment, the LC-MS / MS method for determining polymyxin E1 and E2 in plasma is:

[0039] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0040] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.05, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0041] The chromatographic conditions of the LC-MS / MS are:

[0042] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0043] Gradient elution:

[0044] 0.0-0.5min: A 5%, B 95%; 0.5-3.0min: A95%, B 5%; 3.0-3.5min: A 95%, B5%; 3.5-3.6min: A 5%, B 95%; 3.6-5.0min: A40%, B 60%;

[0045] The flow rate was 0.6 mL / min;

[0046] The column temperature was 45°C;

[0047] Injection volume: 5 μL.

[0048] The mass spectrometry parameters of the LC-MS / MS are:

[0049] Electrospray ion source, positive ion scan mode, ion source temperature: 380°C; spray voltage: 3400 V; evaporation temperature: 380°C; ion transfer tube temperature: 380°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0050] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0051] In one embodiment, the LC-MS / MS method for determining polymyxin E1 and E2 in plasma is:

[0052] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0053] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.35, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0054] The chromatographic conditions of the LC-MS / MS are:

[0055] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0056] Gradient elution: 0.0-0.5 min: A 15%, B 85%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 15%, B 85%; 3.6-5.0 min: A 60%, B 40%;

[0057] The flow rate was 0.4 mL / min; the column temperature was 55°C; and the injection volume was 5 μL.

[0058] The mass spectrometry parameters of the LC-MS / MS are:

[0059] Electrospray ion source, positive ion scan mode, ion source temperature: 420°C; spray voltage: 3600 V; evaporation temperature: 420°C; ion transfer tube temperature: 420°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0060] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] When preparing the mother liquor and working solution of polymyxin E1, E2, and B, the present invention specifically selects a methanol aqueous solution with a methanol content of 20%, which can fully and quickly dissolve the compound and maintain excellent stability. The mother liquor remains stable for 3 months when placed in a -20°C refrigerator, avoiding the disadvantages of long detection time caused by fresh preparation and use, saving time, labor and reagent costs.

[0063] For the detection of polymyxin E1 and E2 provided by the present invention, the linearity of polymyxin E1 was good in the range of 0.058-17.407 μg / mL, and the correlation coefficient R 2 >0.99; Polymyxin E2 has good linearity in the range of 0.100-30.000 μg / mL, with a correlation coefficient R 2 >0.99.

[0064] The pretreatment method of the present invention is simple, adopts protein precipitation and adjusts pH, has high extraction efficiency, and only requires 50 μL of plasma, which is an advantage for clinical sample detection. In addition, the whole blood sample provided meets the requirements of maintaining stability under the conditions of 6 hours at room temperature, 72 days at 4°C, 48 hours at an extract injector (10°C), 57 days at -20°C, and three freeze-thaw cycles at -20°C, greatly meeting the requirements of a uniform process for clinical samples.

[0065] In summary, the method provided by the present invention has simple pretreatment steps, requires a small amount of plasma, can complete the detection of multiple samples in a relatively short time, has high pretreatment efficiency, uses simple reagent preparation, and has very low pretreatment cost. It has low requirements for detection personnel, is highly operational, and is suitable for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1-Figure 3 : Representative chromatograms of polymyxin E in plasma samples

[0067] A: Blank plasma; (1: Polymyxin E1; 2: Polymyxin E2; 3: Polymyxin B)

[0068] B: Blank plasma was spiked with polymyxin E1 (5.802 μg / mL), polymyxin E2 (10.0 μg / mL), and IS (20.0 μg / mL); (1: polymyxin E1; 2: polymyxin E2; 3: polymyxin B)

[0069] C: Plasma sample after IS polymyxin E injection (20.0 μg / mL): (1: polymyxin E1; 2: polymyxin E2; 3: polymyxin B) DETAILED DESCRIPTION

[0070] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited to these embodiments, and the embodiments are only used to illustrate the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0071] 1. Materials

[0072] Drugs: Polymyxin E sulfate (standard: polymyxin E1 content 31.1%, polymyxin E2 content 53.6%), purity: 94.09%, batch number: V-919300-NU1, provided by CFW Laboratories; polymyxin B hydrochloride, purity: 94.8%, batch number: S0703AS, provided by meilunbio.

[0073] Instruments: TSQ Quantis triple quadrupole liquid chromatography-mass spectrometer, ThermoμLtimate 3000UHPLC (WPS584) liquid chromatograph, both are products of Thermo Fisher Scientific, USA.

[0074] 2. Determination method

[0075] Chromatographic conditions:

[0076] The chromatographic column was ThermoAccucore aQ; mobile phase A was methanol + chloride salt; mobile phase B was water (containing 0.5% formic acid);

[0077] Gradient elution:

[0078] 0.0-0.5min: A5-15%, B 85-95%;

[0079] 0.5-3.0min: A95-100%, B 0-5%;

[0080] 3.0-3.5min: A95-100%, B 0-5%;

[0081] 3.5-3.6min: A5-15%, B 85-95%;

[0082] 3.6-5.0min: A40-60%, B 40-60%;

[0083] The flow rate was 0.4-0.6 mL / min; the column temperature was 45-55°C; and the injection volume was 5 μL.

[0084] Mass spectrometry conditions:

[0085] An electrospray ionization source was used in positive ion scan mode; source temperature: 380-420°C; spray voltage: 3400-3600 V; evaporation temperature: 380-420°C; ion transfer tube temperature: 380-420°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; and sweep gas: 10 Arbs. Scanning was performed in multiple reaction monitoring mode, and the monitoring ion transitions were m / z 585.512 → m / z 535.256 (polymyxin E1) at a collision energy of 15.99 eV, m / z 578.462 → m / z 528.489 (polymyxin E2) at a collision energy of 15.95 eV, and m / z 602.675 → m / z 100.971 (internal standard polymyxin B) at a collision energy of 36.26 eV.

[0086] Preparation of control working solution:

[0087] An appropriate amount of polymyxin E sulfate standard was accurately weighed and prepared in 20% methanol to prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 stock solutions. A series of standard working solutions were prepared using a 20% methanol solution with a gradient dilution: polymyxin E1 concentrations of 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, with low, medium, and high quality control concentrations of 1.45, 46.42, and 130.55 μg / mL, respectively. Polymyxin E2 concentrations of 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, with low, medium, and high quality control concentrations of 2.5, 80, and 225 μg / mL, respectively.

[0088] Internal standard solution:

[0089] Accurately weigh an appropriate amount of polymyxin B sulfate standard, prepare a 1000 μg / mL internal standard stock solution with 20% methanol, and then dilute it with 20% methanol to obtain a 200 μg / mL internal standard working solution.

[0090] Preparation of medicated plasma:

[0091] 5 μL of the reference working solution was transferred into 45 μL of blank plasma and vortexed for 20 s to mix. The polymyxin E1 standard curve with mass concentrations of 0.058, 0.116, 0.580, 1.160, 2.901, 5.802, 11.604, and 17.407 μg / mL was obtained. The low, medium, and high concentration quality control concentrations were 0.145, 4.642, and 13.055 μg / mL, respectively. The polymyxin E2 standard curve with mass concentrations of 0.1, 0.2, 1, 2, 5, 10, 20, and 30 μg / mL was obtained. The low, medium, and high concentration quality control concentrations were 0.25, 8.0, and 22.5 μg / mL, respectively.

[0092] Plasma sample processing:

[0093] Add 50 μL of blood sample and 5 μL of internal standard solution to the centrifuge tube in sequence, vortex for 30 seconds, add 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and adjust the pH with protein sodium salt-phosphate, vortex for 2 minutes, centrifuge at 14500 rpm for 10 minutes, take 100 μL of supernatant and add it to the injection bottle, and inject 5 μL.

[0094] Example 1

[0095] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0096] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.15, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0097] The chromatographic conditions of the LC-MS / MS are:

[0098] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0099] Gradient elution: 0.0-0.5 min: A 10%, B 90%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 10%, B 90%; 3.6-5.0 min: A 50%, B 50%;

[0100] The flow rate was 0.5 mL / min; the column temperature was 50°C; and the injection volume was 5 μL.

[0101] The mass spectrometry parameters of the LC-MS / MS are:

[0102] Electrospray ion source, positive ion scan mode, ion source temperature: 400°C; spray voltage: 3500 V; evaporation temperature: 400°C; ion transfer tube temperature: 400°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0103] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0104] Example 2

[0105] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0106] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.05, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0107] The chromatographic conditions of the LC-MS / MS are:

[0108] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0109] Gradient elution:

[0110] 0.0-0.5min: A 5%, B 95%; 0.5-3.0min: A95%, B 5%; 3.0-3.5min: A 95%, B5%; 3.5-3.6min: A 5%, B 95%; 3.6-5.0min: A40%, B 60%;

[0111] The flow rate was 0.6 mL / min;

[0112] The column temperature was 45°C;

[0113] Injection volume: 5 μL.

[0114] The mass spectrometry parameters of the LC-MS / MS are:

[0115] Electrospray ion source, positive ion scan mode, ion source temperature: 380°C; spray voltage: 3400 V; evaporation temperature: 380°C; ion transfer tube temperature: 380°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0116] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0117] Example 3

[0118] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0119] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.35, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0120] The chromatographic conditions of the LC-MS / MS are:

[0121] The chromatographic column was a ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol + 8% potassium chloride (volume ratio 9:1); mobile phase B was water (containing 0.5% formic acid);

[0122] Gradient elution: 0.0-0.5 min: A 15%, B 85%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 15%, B 85%; 3.6-5.0 min: A 60%, B 40%;

[0123] The flow rate was 0.4 mL / min; the column temperature was 55°C; and the injection volume was 5 μL.

[0124] The mass spectrometry parameters of the LC-MS / MS are:

[0125] Electrospray ion source, positive ion scan mode, ion source temperature: 420°C; spray voltage: 3600 V; evaporation temperature: 420°C; ion transfer tube temperature: 420°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0126] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0127] Comparative Example 1

[0128] (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 1.45, 46.42, and 130.55 μg / mL, respectively. The concentrations of polymyxin E2 were 1, 2, 10, 20, 50, 100, 200, and 300 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were 2.5, 80, and 225 μg / mL, respectively.

[0129] (2) Blank plasma, internal standard solution prepared by polymyxin B, and standard working solution were added in sequence, and plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 s, 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and protein sodium salt-phosphate were added to adjust the pH to 7.15, vortexed for 2 min, centrifuged at 14500 rpm for 10 min, 100 μL of supernatant was taken and added to the injection bottle, 5 μL was injected, and the pretreated solution was analyzed by LC-MS / MS;

[0130] The chromatographic conditions of the LC-MS / MS are:

[0131] The chromatographic column was ThermoAccucore aQ (50 mm × 2.1 mm, 2.6 μm); mobile phase A was methanol; mobile phase B was water (containing 0.5% formic acid);

[0132] Gradient elution: 0.0-0.5 min: A 10%, B 90%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 10%, B 90%; 3.6-5.0 min: A 50%, B 50%;

[0133] The flow rate was 0.5 mL / min; the column temperature was 50°C; and the injection volume was 5 μL.

[0134] The mass spectrometry parameters of the LC-MS / MS are:

[0135] Electrospray ion source, positive ion scan mode, ion source temperature: 400°C; spray voltage: 3500 V; evaporation temperature: 400°C; ion transfer tube temperature: 400°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; purge gas: 10 Arbs.

[0136] (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

[0137] After comparing the method of Example 1, it was found that the retention time of this detection method was 8 minutes, and the lower limit of quantitative detection was 0.426 μg / ml. Compared with the detection methods of Examples 1-2 of the present invention, the analysis time was longer and the sensitivity was poorer. The detection time of the methods of Examples 1-3 of the present invention was only 5 minutes, and the lower limit of quantitative detection was 0.158 μg / ml.

[0138] 3. Methodological Investigation

[0139] Exclusive properties:

[0140] 50 μL of blank plasma from six different sources was sampled and analyzed according to the "Plasma Sample Processing" procedure to obtain blank plasma chromatograms. Another aliquot of blank plasma was spiked with the analytes (polymyxin E1 and E2) and internal standard, and another aliquot of actual plasma from a patient after administration was processed in the same manner to obtain the corresponding chromatograms.

[0141] Standard curve and lower limit of quantification:

[0142] 45 μL of blank plasma was accurately drawn, and 5 μL of polymyxin E standard solution of different concentrations was added according to the concentration range of different standard curves to form a series of standard curve samples. According to the "plasma sample processing" operation, HPLC-MS / MS analysis was performed, with the known concentration as the horizontal axis and the ratio of the peak area of polymyxin E at different concentrations to the peak area of the internal standard as the vertical axis. The weighted least squares method (weight coefficient is 1 / X 2 ) to perform regression operation and obtain the linear regression equation.

[0143] Precision and extraction recovery:

[0144] Six replicates of human plasma samples containing low, medium, and high quality control concentrations of polymyxin E were prepared in parallel. Intra- and inter-day precision was evaluated using the procedures described in "Plasma Sample Processing" for at least three analytical runs over at least two days, with at least six measurements at each concentration. Six replicates of human plasma quality control samples containing low, medium, and high polymyxin E concentrations were prepared in parallel, following the procedures described in "Plasma Sample Processing" to obtain the target analyte area, A1. Samples corresponding to the polymyxin E quality control concentrations were prepared from the supernatant extracted from blank plasma samples. After vortexing and centrifugation, the peak area, A2, was obtained by injection. Six replicates of each concentration were measured, and the extraction recovery was A1 / A2 × 100%.

[0145] Matrix Effects:

[0146] Matrix-based samples were prepared by adding 45 μL of each of six blank plasmas from different sources, one high-fat plasma, and one hemolyzed plasma. Add 25 μL of 0.1 M ZnSO₄ and 100 μL of acetonitrile, vortex for 2 minutes, and centrifuge at 14,500 rpm for 10 minutes. Matrix-based samples were then prepared by adding 5 μL of low-, medium-, and high-mass concentrations of polymyxin E working solution and 5 μL of internal standard. After injection, the peak area ratio of the analyte to the internal standard, A3, was calculated. The plasma was replaced with ultrapure water and the same procedure was repeated to obtain the analyte, A4. The internal standard normalized matrix factor was calculated as A3 / A4 × 100%.

[0147] stability:

[0148] Low, medium, and high concentration plasma quality control samples were prepared and tested for stability after 6 hours at room temperature, 7 days at 4°C, 48 hours at the extract injector (10°C), 57 days at -20°C, and three freeze-thaw cycles at -20°C. The stability of the working solution was also tested after 6 hours at room temperature and 7 days at -20°C.

[0149] 4. Results

[0150] 4.1 Methodological Evaluation of Example 1

[0151] 4.1.1 Specificity

[0152] The retention times of polymyxin E1, polymyxin E2, and polymyxin B were approximately 2.31 min, 2.18 min, and 2.35 min, respectively. Endogenous substances in plasma did not interfere with the determination of target analytes and internal standards, and the specificity was good. Figure 1 .

[0153] 4.1.2 Standard curve and lower limit of quantification:

[0154] The linear relationship between polymyxin E1 and E2 was good in the range of 0.058-17.407 μg / mL and 0.100-30.000 μg / mL, respectively. The standard curve equations were Y=0.00374143+0.213039*X(R 2 =0.9981) and Y = 0.000267707 + 0.206645 * X (R 2 =0.9973).

[0155] 4.1.3 Precision and recovery

[0156] The intra- and inter-batch precisions of the quality control at low, medium, and high concentrations of polymyxin E1 and E2 were 1.030%-11.538%, the extraction recoveries were 78.606%-85.007%, and the intra- and inter-batch precisions of the lower limit of quantification were less than 20%, all meeting the method validation requirements (Table 1).

[0157] Table 1 Precision of polymyxin E1 and polymyxin E2 in plasma (n=18)

[0158]

[0159] 4.1.4 Matrix Effects

[0160] The matrix effects of polymyxin E1 at low, medium, and high mass concentrations after internal standard correction were 93.1%, 97.0%, and 96.1%, respectively. The matrix effects of polymyxin E2 were 97.2%, 100.3%, and 97.3%, respectively. The RSDs were between 2.619% and 8.724%, indicating that the matrix effect did not affect the detection.

[0161] 4.2 Study on the stability of plasma pretreatment methods

[0162] Example 4

[0163] Plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 seconds, 25 μL of 0.1 mol ZnSO4 solution and 100 μL of acetonitrile were added, and the pH was adjusted to 7.15 with protein sodium salt-phosphate. The tube was vortexed for 2 minutes, centrifuged at 14500 rpm for 10 minutes, and 100 μL of supernatant was added to the injection bottle. This method only requires 50 μL of plasma.

[0164] Example 5

[0165] Plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 seconds, 25 μL of 0.1 mol ZnSO4 solution and 100 μL of acetonitrile were added, and the pH was adjusted to 7.05 with protein sodium salt-phosphate. The tube was vortexed for 2 minutes, centrifuged at 14500 rpm for 10 minutes, and 100 μL of the supernatant was added to the injection bottle. This method only requires 50 μL of plasma.

[0166] Example 6

[0167] Plasma pretreatment was performed by protein precipitation method: 50 μL of blood sample and 5 μL of internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 seconds, 25 μL of 0.1 mol ZnSO4 solution and 100 μL of acetonitrile were added, and the pH was adjusted to 7.35 with protein sodium salt-phosphate. The tube was vortexed for 2 minutes, centrifuged at 14500 rpm for 10 minutes, and 100 μL of supernatant was added to the injection bottle. This method only requires 50 μL of plasma.

[0168] Comparative Example 2

[0169] Plasma pretreatment by protein precipitation: Add blood sample and internal standard to a centrifuge tube, vortex for 30 seconds, add 0.1 mol ZnSO4 and acetonitrile, vortex for 2 minutes, and centrifuge at 14,500 rpm for 10 minutes. This method requires 100 μL of plasma.

[0170] Comparative Example 3

[0171] Plasma pretreatment was performed by protein precipitation method: blood sample and internal standard solution were added to the centrifuge tube in sequence, vortexed for 30 seconds, 0.1 mol ZnSO4 solution, acetonitrile, and phosphate were added to adjust the pH to 7.45, vortexed for 2 minutes, centrifuged at 14500 rpm for 10 minutes, and 100 μL of the supernatant was added to the injection bottle. The plasma dosage required for this method was 80 μL.

[0172] The plasma quality control samples of Examples 4-6 were stable under all conditions of 6 h at room temperature, 72 d at 4 ° C, 48 h at the extract injector (10 ° C), 57 d at -20 ° C, and 3 freeze-thaw cycles at -20 ° C, with RSDs less than 7.978%; the working solution was stable at room temperature for 6 h and 7 d at -20 ° C, with REs less than 9.300%, and the amount of plasma used in the analysis samples was small, which can greatly improve the extraction efficiency.

[0173] Table 2 pH and partial anticoagulant factors of pre-treated plasma in each embodiment and comparative example

[0174]

[0175]

[0176] The results in Table 2 show that the plasma pretreatment method of the present invention uses a protein precipitation method to adjust the plasma pH range to 7.05-7.35 using protein sodium salt-phosphate, and the coagulation factors change little or remain basically unchanged and are basically stable within 48 hours.

Claims

1. A method for detecting polymyxin E1 and E2 in plasma by LC-MS / MS, characterized in that: The following steps are involved: (1) Prepare a series of standard working solutions including polymyxin E1 and E2 by gradient dilution using 20% methanol solution; (2) Blank plasma, an internal standard solution prepared from polymyxin B, a standard working solution, and a protein precipitation method were sequentially added to perform plasma pretreatment, and the pretreated solution was analyzed using LC-MS / MS; the chromatographic conditions of the LC-MS / MS were as follows: The chromatographic column was a Thermo Accucore aQ 50 mm × 2.1 mm, 2.6 μm; Mobile phase A: methanol + 8% potassium chloride, volume ratio 9:1; mobile phase B: water containing 0.5% formic acid; Gradient elution: 0.0-0.5 min: A 5-15%, B 85-95%; 0.5-3.0 min: A 95-100%, B 0-5%; 3.0-3.5 min: A 95-100%, B 0-5%; 3.5-3.6 min: A 5-15%, B 85-95%; 3.6-5.0 min: A 40-60%, B 40-60%; Flow rate 0.4-0.6 mL / min; Column temperature is 45-55°C; Injection volume: 5 μL; (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

2. The method according to claim 1, characterized in that The chromatographic conditions of the LC-MS / MS are: The chromatographic column was a Thermo Accucore aQ 50 mm × 2.1 mm, 2.6 μm; Mobile phase A: methanol + 8% potassium chloride, volume ratio 9:1; mobile phase B: water containing 0.5% formic acid; Gradient elution: 0.0-0.5 min: A 10%, B 90%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 10%, B 90%; 3.6-5.0 min: A 50%, B 50%; The flow rate was 0.5 mL / min; The column temperature was 50 °C; Injection volume: 5 μL.

3. The method according to claim 1, characterized in that The mass spectrometry parameters of the LC-MS / MS are: Electrospray ionization source, positive ion scan mode, ion source temperature: 380-420°C; spray voltage: 3400-3600 V; evaporation temperature: 380-420°C; ion transfer tube temperature: 380-420°C; sheath Air: 40 Arbs; Heating auxiliary gas: 20 Arbs; Purge gas: 10 Arbs.

4. The method according to claim 3, characterized in that The mass spectrometry parameters of the LC-MS / MS are: Electrospray ionization source, positive ion scan mode, ion source temperature: 400°C; spray voltage: 3500 V; evaporation temperature: 400°C; ion transfer tube temperature: 400°C; sheath gas: 40 Arbs; Heating auxiliary gas: 20 Arbs; Purge gas: 10 Arbs.

5. The method according to claim 1, wherein The plasma pre-treatment method is: Add blood sample and internal standard solution to the centrifuge tube in sequence, vortex, add ZnSO4 solution and acetonitrile, add buffer, vortex, centrifuge, and take the supernatant.

6. The method according to claim 5, characterized in that The method for the plasma pretreatment is: Add 50 μL of blood sample and 5 μL of internal standard solution to the centrifuge tube in sequence, vortex for 30 s, add 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, and adjust the pH with protein sodium salt-phosphate, vortex for 2 min, centrifuge at 14500 rpm for 10 min, take 100 μL of supernatant and add it to the injection bottle, and inject 5 μL.

7. The method according to claim 6, characterized in that The pH is 7.05-7.

35.

8. The method according to claim 7, characterized in that The pH was 7.

15.

9. The method according to any one of claims 1 to 8, characterized in that The following steps are involved: (1) Accurately weigh an appropriate amount of polymyxin E sulfate standard and prepare 580 μg / mL polymyxin E1 and 1000 μg / mL polymyxin E2 standard stock solutions using 20% methanol as solvent. A series of standard working solutions were prepared by gradient dilution with 20% methanol solution. The concentrations of polymyxin E1 were 0.58, 1.16, 5.80, 11.60, 29.01, 58.02, 116.04, and 174.07 μg / mL, respectively. The low, medium, and high concentration quality control concentrations were: 1.45, 46.42, 130.55 ug / mL; polymyxin E2 concentrations were 1, 2, 10, 20, 50, 100, 200, 300 ug / mL, and the low, medium, and high concentration quality control concentrations were 2.5, 80, 225 ug / mL respectively; (2) Add blank plasma, internal standard solution prepared by polymyxin B, and standard working solution in sequence, and perform plasma pretreatment by protein precipitation method: add 50 μL of blood sample and 5 μL of internal standard solution to the centrifuge tube in sequence, vortex for 30 s, add 25 μL of 0.1 mol ZnSO4 solution, 100 μL of acetonitrile, adjust the pH to 7.15 with protein sodium salt-phosphate, vortex for 2 min, centrifuge at 14500 rpm for 10 min, take 100 μL of supernatant and add it to the injection bottle, inject 5 μL, and analyze the pretreated solution using LC-MS / MS; The chromatographic conditions of the LC-MS / MS are: The chromatographic column was a Thermo Accucore aQ 50 mm × 2.1 mm, 2.6 μm column; mobile phase A was methanol + 8% potassium chloride, with a volume ratio of 9:1; mobile phase B was water containing 0.5% formic acid; Gradient elution: 0.0-0.5 min: A 10%, B 90%; 0.5-3.0 min: A 100%, B 0%; 3.0-3.5 min: A 100%, B 0%; 3.5-3.6 min: A 10%, B 90%; 3.6-5.0 min: A 50%, B 50%; The flow rate was 0.5 mL / min; the column temperature was 50 °C; the injection volume was 5 μL; The mass spectrometry parameters of the LC-MS / MS were as follows: ion source temperature: 400°C; spray voltage: 3500 V; evaporation temperature: 400°C; ion transfer tube temperature: 400°C; sheath gas: 40 Arbs; heating auxiliary gas: 20 Arbs; Purge Air: 10 Arbs; (3) A standard curve was established with the ratio of the chromatographic peak area of polymyxin E1 and E2 to the internal standard polymyxin B as the ordinate and the polymyxin E1 and E2 in human blank plasma as the abscissa. The concentrations of polymyxin E1 and E2 in the human plasma samples to be tested were calculated based on the standard curve.

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