A method for simultaneously detecting the content of polymyxin and caspofungin and application of internal standard

Abstract

The present invention relates to the field of pharmaceutical detection technology, in particular to a method for simultaneously detecting the content of polymyxin and caspofungin, an internal standard and its application. The method for simultaneously detecting polymyxin B1, B2, E1, E2 and caspofungin in blood by high performance liquid chromatography tandem mass spectrometry comprises: preparing a standard curve equation, processing to obtain a sample to be tested, and detecting the sample to be tested. The method of the present invention uses bacitracin zinc as an internal standard, adopts a method of simultaneous detection after mixing with a standard product, and effectively improves the detection efficiency of polymyxin B1, B2, E1, E2 and caspofungin. The detection method of the present invention has good reproducibility, small matrix effect, and improves the accuracy of the detection result. The present invention establishes a method for simultaneously detecting polymyxin B1, B2, E1, E2 and caspofungin, the detection process is simple and fast, the experimental cost is reduced, the analysis time is short, and it is more conducive to large-throughput sample detection.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical detection technology, and in particular to a method for simultaneously detecting the contents of polymyxin and caspofungin, an internal standard substance and application thereof. Background Art

[0002] Polymyxin (PM) is a general term for a group of basic polypeptide antibiotics isolated from Paenibacillus polymyxa, mainly including five varieties, namely: A, B, C, D and E. At present, there are only two types of polymyxin drugs in clinical use: polymyxin B (mainly composed of polymyxin B1 and polymyxin B2) and polymyxin E (composed of polymyxin E1 and polymyxin E2, also known as colistin). Polymyxin mainly exerts its antibacterial effect by binding to the lipopolysaccharide of the outer membrane of Gram-negative bacteria and destroying the cell membrane. It has antibacterial effects on multidrug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Since both polymyxin B and polymyxin E have serious adverse reactions in clinical applications, therapeutic drug monitoring is required for both drugs in clinical applications to determine the clinical dosage.

[0003] Caspofungin (CPF) is a semisynthetic lipopeptide compound of the echinocandin class of drugs, which is now widely used to treat invasive fungal infections, especially in patients with hematological malignancies and hematopoietic stem cell transplant recipients. Caspofungin mainly targets the β-actin on the fungal cell wall. -1 ,3-D-glucan synthase, inhibiting the synthesis of fungal cell walls, leading to the death of fungal cells. This unique mechanism of action makes caspofungin antibacterial against a variety of fungi, including Candida, Aspergillus, and some fungi resistant to azole drugs. Its main adverse reactions are allergies, such as rash, facial swelling, itching, warmth, or bronchospasm.

[0004] Patients with clinical critical infections often face multidrug-resistant bacterial infections. Some patients have open wounds due to falls or abrasions, which increases the risk of infection. At the same time, some critically ill patients have poor immunity due to long-term hospitalization, which greatly increases the risk of nosocomial cross-infection. For patients with bacterial and fungal infections, clinical drug options are very limited. In particular, for Gram-negative bacteria resistant to carbapenems, there are currently no other targeted antibiotics available except polymyxin B or E. For patients with combined fungal infections, caspofungin, as a new generation of antifungal drugs, has significant advantages in fungal infection control. In clinical practice, for patients with multidrug-resistant bacteria and fungal infections, polymyxins and caspofungin are often used in combination to effectively control multiple infections. Therefore, it is a very necessary measure in the clinical treatment process to carry out simultaneous testing of polymyxins and caspofungin to quickly identify whether the blood drug concentration meets the standard so as to adjust the dosage in time to effectively control the infection.

[0005] At present, the method commonly used to determine the content of polymyxin B1, B2, E1, E2 and caspofungin in the blood is high performance liquid chromatography tandem mass spectrometry. However, there is currently no method that can detect these three drugs simultaneously. Peptide drugs have large molecular weights, are not artificially synthesized, and have no available isotope-labeled forms. In therapeutic drug monitoring, the determination of blood concentrations of peptide drugs usually faces the challenge of selecting internal standard compounds, and the existing liquid chromatography-mass spectrometry (LC-MS) research on polymyxin B1, B2, E1, E2 and caspofungin has the disadvantages of cumbersome operation, poor sensitivity, narrow linear range and long detection time in practical applications. Some literatures have reported methods of using polymyxin B and E as internal standards for each other. However, in clinical practice, due to insufficient supply of the two drugs, they often replace each other, resulting in the possibility that polymyxin B and polymyxin E may exist in the patient's body at the same time. The method of using polymyxin B or E as internal standards for each other is not applicable to such situations.

[0006] Therefore, it is necessary to propose a liquid chromatography-mass spectrometry analysis method for simultaneously detecting polymyxin B1, B2, E1, E2 and caspofungin which is simple, accurate and time-saving. To this end, the present invention proposes a method for simultaneously detecting the contents of polymyxin and caspofungin, an internal standard and an application thereof. Summary of the invention

[0007] The present invention provides a method for simultaneously detecting the contents of polymyxin B1, B2, E1, E2 and caspofungin, an internal standard and an application thereof, so as to at least solve the problems in the prior art of the blood drug concentration detection method of polymyxin B1, B2, E1, E2 and caspofungin that the pretreatment operation is relatively complicated, the quantitative result has poor accuracy, and the analysis time is relatively long, or at least solve the problem that there is currently no method capable of simultaneously detecting the contents of polymyxin B1, B2, E1, E2 and caspofungin in blood.

[0008] In order to achieve the above object, according to one aspect of the present invention, a method for simultaneously detecting the content of polymyxin and caspofungin is provided, comprising:

[0009] S1. Prepare the standard curve equation, including:

[0010] Prepare standard working solutions and internal standard working solutions with gradient concentrations; prepare polymyxin reference substance and caspofungin reference substance into standard working solutions, and use bacitracin zinc as an internal standard to prepare internal standard working solutions;

[0011] The standard working solution and plasma are mixed to obtain a drug-containing plasma standard solution, the internal standard working solution and the protein precipitant are added to the drug-containing plasma standard solution, mixed and centrifuged, the supernatant is taken and diluted to obtain a standard solution to be tested, the supernatant and the diluent are mixed and diluted at a volume ratio of 1:2, the standard solution to be tested is detected by high performance liquid chromatography-mass spectrometry, and a standard curve equation is obtained;

[0012] S2. Processing to obtain a sample to be tested, including: adding an internal standard working solution and a protein precipitant to the sample to be tested, mixing and centrifuging, taking a supernatant and diluting to obtain a sample to be tested, and mixing and diluting the supernatant and the diluent at a volume ratio of 1:2;

[0013] S3. Testing of the test samples, including: using a high performance liquid chromatography-mass spectrometer to test the test samples, substituting the test results into a standard curve equation and calculating to obtain the concentrations of polymyxin and caspofungin in the test samples.

[0014] Further, in S1, polymyxins include polymyxin B1, B2, E1 and E2, the solvent of the standard working solution includes a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the concentration of the polymyxin B1 reference substance is 1.56 mg·L -1 ~100.00mg·L -1 The concentration of polymyxin B2 reference substance is 0.14 mg·L -1 ~8.78mg·L -1 The concentration of polymyxin E1 reference substance was 0.44 mg·L -1 ~27.88mg·L -1 The concentration of polymyxin E2 reference substance is 0.78 mg·L -1 ~50.00mg·L -1 The concentration of caspofungin reference substance was 1.56 mg·L -1 ~100.00mg·L -1 The solvent of the internal standard working solution includes a formic acid aqueous solution with a volume percentage concentration of 0.1 to 0.3%, and the concentration of bacitracin zinc in the internal standard working solution is 5 to 10 mg·L -1 .

[0015] Furthermore, in S1, the volume ratio of the standard working solution and the plasma is 5-10:90-95; the conditions for mixing the standard working solution and the plasma include: vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 min, and the plasma is mammalian plasma, such as pig and cow plasma.

[0016] Furthermore, in S1, the protein precipitant is acetonitrile; the volume ratio of the drug-containing plasma standard solution, the internal standard working solution and the protein precipitant is 80-120:5-10:200-300, and the mixing conditions of the drug-containing plasma standard solution, the internal standard working solution and the protein precipitant include: vortex mixing at a rotation speed of 1200-1800rpm for 0.5-1min; the centrifugation conditions include: centrifugation at a rotation speed of 12500-13500rpm for 3-5min; the diluent of the supernatant is a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the supernatant and the diluent are mixed and diluted at a volume ratio of 1:2.

[0017] Here, other precipitants cannot achieve the effect of detecting three drugs at the same time. Commonly used precipitants, including methanol, acetonitrile, and trichloroacetic acid, were used in the test. Only acetonitrile can achieve the effect of this method. Trichloroacetic acid can decompose caspofungin; methanol requires a 1:4 precipitation ratio (acetonitrile is 1:2, trichloroacetic acid is 1:1), which reduces the final detection concentration by 2 times, the response value is poor, and the low concentration point cannot be accurately quantified, thus limiting the detection concentration range.

[0018] Further, in S2, the volume ratio of the sample to be tested, the internal standard working solution and the protein precipitant is 80-120:5-10:200-300.

[0019] Further, in S2, the conditions for mixing the sample to be tested, the internal standard working solution and the protein precipitant include: vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 min; the conditions for centrifugation include: centrifugation at a rotation speed of 12500-13500 rpm for 3-5 min; the diluent for the supernatant is a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the supernatant and the diluent are mixed and diluted at a volume ratio of 1:2.

[0020] Further, in S1 and S3, the parameter settings of the high performance liquid chromatography mass spectrometer include: the high performance liquid chromatography uses a reverse phase chromatography column C18 column, and the particle size of the C18 column is 1.8 to 3.8 μm;

[0021] Mobile phase: Phase A: aqueous solution containing 0.1-0.2% (v / v) formic acid; Phase B: acetonitrile;

[0022] Flow rate: 0.25-0.35 mL / min; column temperature: 25-30°C; injection volume: 5-10 μL; analysis time: 3.0-4.0 min.

[0023] Further, the elution conditions of high performance liquid chromatography are:

[0024] 0~1.00min, phase A: 5~90%; phase B: 10~95%;

[0025] 1.01~1.50min, phase A: 5~15%; phase B: 85~95%;

[0026] 1.51~2.00min, phase A: 5~90%; phase B: 10~95%;

[0027] 2.01~3.50min, phase A: 85~95%; phase B: 5~15%.

[0028] Here, the setting of elution conditions is mainly to optimize the peak shape and retention time of the chromatographic peak to achieve an analysis speed of 3-4 minutes.

[0029] Furthermore, in S1 and S3, the parameter settings of the high performance liquid chromatography-mass spectrometer include: the detection conditions of the mass spectrometer include: capillary temperature: 270-320°C; electrospray voltage: 3000-3500V; vaporization temperature 100-150°C; sheath gas flow rate: 9-12L / min; auxiliary gas flow rate: 1.5-4.5L / min; lens voltage: 55-75V; collision energy: 10-30V.

[0030] The second aspect of the present invention proposes the use of bacitracin zinc as an internal standard in detecting the content of polymyxin and / or caspofungin.

[0031] The third aspect of the present invention provides an internal standard for detecting the content of polymyxin and / or caspofungin, including bacitracin zinc.

[0032] The beneficial effects of the technical solution of the present invention are:

[0033] 1. The present invention innovatively utilizes bacitracin zinc as an internal standard and adopts a method of simultaneous detection after mixing standard products, thereby effectively improving the detection efficiency of polymyxin B1, B2, E1, E2 and caspofungin.

[0034] 2. The detection method of the present invention uses acetonitrile as a protein precipitant to achieve a technical effect in which the matrix effect meets the requirements.

[0035] 3. The detection method of the present invention is that in the blood, polymyxin B1 is 0.156 mg·L -1 To 10.00mg·L -1In the range of 0.014 mg·L -1 To 0.878mg·L -1 In the range of 0.044 mg·L -1 To 2.788mg·L -1 In the range of 0.078 mg·L -1 To 5.00mg·L -1 In the range of 0.156 mg·L -1 To 10.00mg·L -1 The linearity is good within the range, and the correlation coefficient R 2 ﹥0.9900.

[0036] 4. The detection method of the present invention has good reproducibility, the matrix effect meets the requirements, and the accuracy of the test results is improved. A method for simultaneously detecting polymyxin B1, B2, E1, E2 and caspofungin is established, which makes the detection process simple and fast, reduces the experimental cost, shortens the analysis time, and is more conducive to large-throughput sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the chromatogram of polymyxin B1 in the standard solution in Example 2;

[0038] Figure 2 is the chromatogram of polymyxin B2 in the standard solution in Example 2;

[0039] Figure 3 is the chromatogram of polymyxin E1 in the standard solution in Example 2;

[0040] Figure 4 is the chromatogram of polymyxin E2 in the standard solution in Example 2;

[0041] Figure 5 is the chromatogram of caspofungin in the standard solution in Example 2;

[0042] Figure 6 The chromatogram of the internal standard compound bacitracin zinc in the standard solution in Example 2;

[0043] Figure 7 The chromatogram of polymyxin B1 in the spiked plasma sample in Example 2;

[0044] Figure 8 The chromatogram of polymyxin B2 in the spiked plasma sample in Example 2;

[0045] Fig. 9 The chromatogram of polymyxin E1 in the spiked plasma sample in Example 2;

[0046] Fig.10The chromatogram of polymyxin E2 in the spiked plasma sample in Example 2;

[0047] Fig.11 The chromatogram of caspofungin in the spiked plasma sample in Example 2;

[0048] Fig.12 The chromatogram of the internal standard compound bacitracin zinc in the spiked plasma sample in Example 2;

[0049] Fig.13 The chromatograms of polymyxin B1, B2, E1, E2 and caspofungin in the standard solution in Experimental Example 2;

[0050] Fig.14 The chromatograms of polymyxin B1, B2, E1, E2 and caspofungin in spiked plasma samples in Experimental Example 2; DETAILED DESCRIPTION

[0051] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0052] The embodiment of the present invention provides a method for simultaneously detecting the content of polymyxin and caspofungin, and the method comprises at least the following steps:

[0053] S1. Prepare the standard curve equation, including:

[0054] Prepare standard working solutions and internal standard working solutions with gradient concentrations; Bacitracin zinc is used as the internal standard in the internal standard working solution; the standard working solution contains polymyxin B1, B2, E1, E2 and caspofungin reference substances;

[0055] The standard working solution and blank plasma were mixed to obtain drug-containing plasma standard solutions containing different concentrations of polymyxin B1, B2, E1, E2 and caspofungin;

[0056] Adding internal standard working solution and protein precipitant to the drug-containing plasma standard solution; after mixing and centrifugation, taking the supernatant and mixing it with formic acid aqueous solution in a ratio of 1:2 to obtain the standard solution to be tested;

[0057] The standard solution to be tested is tested by high performance liquid chromatography-mass spectrometry to obtain a standard curve equation;

[0058] S2, processing to obtain a sample to be tested, including: taking the blood to be tested, adding an internal standard working solution and a protein precipitant; after mixing and centrifuging, taking the supernatant and mixing it with a formic acid aqueous solution in a ratio of 1:2 to obtain a sample to be tested;

[0059] S3. Detecting the sample to be tested, including: using a high performance liquid chromatography-mass spectrometer to detect the sample to be tested, substituting the test result into a standard curve equation, and obtaining the concentrations of polymyxin B1, B2, E1, E2 and caspofungin in the sample to be tested.

[0060] The present invention adopts formic acid aqueous solution to prepare standard solution, uses mammalian plasma (such as pig, cattle, etc.) as blank plasma, and selects acetonitrile as protein precipitant to ensure that pretreatment is performed without isotope internal standard, thereby simplifying the steps of pretreatment operation under the premise of meeting detection requirements.

[0061] As a preferred technical solution of an embodiment of the present invention, the formic acid aqueous solution is a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3% (hereinafter referred to as a diluent). The concentration of formic acid water will have a certain effect on the stability of polymyxin B1, B2, E1, E2 and caspofungin, so a formic acid aqueous solution with a volume percentage concentration of 0.2% is more preferred. The use of this diluent can not only ensure the solubility of polymyxin B1, B2, E1, E2 and caspofungin, but also ensure the stability of polymyxin B1, B2, E1, E2 and caspofungin.

[0062] As a specific implementation, the preparation of the standard working solution can be carried out by the following method:

[0063] (1) Using the polymyxin B standard, prepare the mother solution A with the above dilution. The concentration of the mother solution A can be 200-400 mg·L -1 ;

[0064] (2) Using the polymyxin E standard, prepare the mother solution B with the above dilution. The concentration of the mother solution B can be 200-400 mg·L -1 ;

[0065] (3) Using caspofungin standard, prepare mother solution C with the above diluent. The concentration of mother solution C can be 200-400 mg·L -1 ;

[0066] (4) Mix mother solution A, mother solution B and mother solution C at the same concentration and volume ratio of 2:1:2, and dilute with the above diluent. The concentration of polymyxin B1 in the obtained standard working solution can be 100-200 mg·L -1 , preferably 100 mg·L -1 ; The concentration of polymyxin E2 can be 50-100 mg·L -1 , preferably 50 mg·L -1 The concentration of caspofungin can be 100-200 mg·L -1 , preferably 100 mg·L -1 , which is convenient for subsequent preparation;

[0067] (4) diluting with the above diluent to prepare solutions with seven concentration gradients, specifically, the concentrations of polymyxin B1, B2, E1, E2 and caspofungin solutions are shown in Table 1;

[0068] Table 1 Standard working concentrations of polymyxin B1, B2, E1, E2 and caspofungin

[0069]

[0070]

[0071] As a specific embodiment, the preparation of the drug-containing plasma standard solution can be carried out by the following method:

[0072] (1) Use mammalian blank plasma, such as pig and cow, as a simulated human blank plasma.

[0073] (2) The seven concentration gradient standard working solutions containing polymyxin B1, B2, E1, E2 and caspofungin obtained after dilution were mixed with blank plasma at a volume ratio of 10:90, and the mixing conditions were vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 minute, preferably vortex mixing at 1400-1600 rpm for 1 minute to obtain drug-containing plasma standard solutions. Specifically, the concentrations of drug-containing plasma standard solutions of polymyxin B1, B2, E1, E2 and caspofungin can be shown in Table 2, and stored at -20°C.

[0074] Table 2 Concentrations of standard plasma solutions of polymyxin B1, B2, E1, E2 and caspofungin

[0075]

[0076] As a preferred technical solution of the embodiment of the present invention, the internal standard working solution is prepared by using the formic acid aqueous solution (hereinafter referred to as diluent), and the volume percentage concentration of formic acid is preferably 0.2%. The use of this solvent can not only ensure the solubility of the internal standard substance (bacitracin zinc), but also ensure the stability of the internal standard substance (bacitracin zinc).

[0077] In order to further improve the stability, the diluent used a formic acid aqueous solution with a volume percentage concentration of 0.2%, and the concentration of bacitracin zinc in the internal standard working solution was 5-10 mg·L -1 , preferably 8 mg·L -1 .

[0078] As a specific implementation method, the preparation of the internal standard working solution can be carried out by the following method:

[0079] (1) Prepare a standard stock solution of bacitracin zinc with the above dilution at a concentration of 100-200 mg / L -1 , preferably 100 mg·L -1 .

[0080] (2) Dilute with the above diluent, the concentration of bacitracin zinc in the obtained standard internal standard solution can be 5 to 10 mg·L -1 , preferably 8 mg·L -1 The internal standard working solution was stored at -20°C.

[0081] As a preferred technical solution of an embodiment of the present invention, in S1, the volume ratio of the drug-containing plasma standard solution, the internal standard working solution and the protein precipitant is 80-120:5-10:200-300, preferably 100:10:200.

[0082] As a preferred technical solution of an embodiment of the present invention, in S1, the mixing condition is vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 minute, preferably vortex mixing at a rotation speed of 1400-1600 rpm for 1 minute; the centrifugal condition is centrifugation at a rotation speed of 12500-13500 rpm for 3-5 minutes, preferably centrifugation at a rotation speed of 13000 rpm for 3 minutes, and the supernatant is diluted with the diluent at a ratio of 1:2 for detection.

[0083] As a preferred technical solution of an embodiment of the present invention, in S1, the obtained standard solutions of polymyxin B1, B2, E1, E2, caspofungin and internal standard with multiple different concentrations are detected by high performance liquid chromatography-mass spectrometry, and the peak areas of polymyxin B1, B2, E1, E2, caspofungin and internal standard are obtained from the chromatograms of the standard solutions of polymyxin B1, B2, E1, E2, caspofungin and internal standard, respectively. The ratios of the peak areas of the polymyxin B1, B2, E1, E2 and caspofungin standard solutions with the peak areas of the internal standard are used as the ordinate y1 of the standard curve equation, and the ratios of the concentrations of the polymyxin B1, B2, E1, E2 and caspofungin standard solutions with the concentrations of the internal standard working solution are used as the abscissa x1 of the standard curve equation. The data of the multiple different concentrations obtained by the above detection are subjected to linear regression, and the standard curve equation is fitted to be y=a*x+b, and the linear equation coefficients a and b are obtained.

[0084] As a preferred technical solution of an embodiment of the present invention, the volume ratio of the blood to be tested, the internal standard working solution and the protein precipitant is 80-120:5-10:200-300, preferably 100:10:200.

[0085] As a preferred technical solution of an embodiment of the present invention, in S2, the mixing condition is vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 minute, preferably vortex mixing at a rotation speed of 1400-1600 rpm for 1 minute; the centrifugal condition is centrifugation at a rotation speed of 12500-13500 rpm for 3-5 minutes, preferably centrifugation at a rotation speed of 13000 rpm for 3 minutes, and the supernatant is diluted with the diluent at a ratio of 1:2 for detection.

[0086] As a preferred technical solution of an embodiment of the present invention, in S3, a high performance liquid chromatography-mass spectrometer is used to detect the sample to be tested obtained in the above step S2, and a chromatogram of polymyxin B1, B2, E1, E2, caspofungin and the internal standard of the sample to be tested is obtained. From the chromatogram of polymyxin B1, B2, E1, E2, caspofungin and the internal standard, the peak surface of polymyxin B1, B2, E1, E2, caspofungin and the internal standard can be obtained. The ratio of the peak areas of polymyxin B1, B2, E1, E2, caspofungin and the internal standard y1 is substituted into the standard curve equation y=a*x+b in the above step S1, and the relative concentration x of polymyxin B1, B2, E1, E2, caspofungin and the internal standard in the test sample is obtained by calculation. The concentration of the internal standard working solution is known, and the concentrations of polymyxin B1, B2, E1, E2 and caspofungin in the test blood sample are calculated.

[0087] As a preferred technical solution of an embodiment of the present invention, in S1 and S3, the high performance liquid chromatography adopts a C18 reverse phase chromatographic column; the particle size of the reverse phase chromatographic column adopted is 1.8-3.8 μm; and preferably 3.5 μm, the inner diameter of the reverse phase chromatographic column adopted is preferably 2.1-3.0 mm, and the length of the chromatographic column is preferably 100 mm; specific columns that can be selected include: ThermoHypersil GOLD C18 2.1×100mm, 1.9μm; Waters CORTECS T3 2.1×100mm, 2.7μm; ThermoHypersil GOLD C18 2.1×100mm, 3.5μm, etc.

[0088] As a preferred technical solution of an embodiment of the present invention, the mobile phase of the high performance liquid chromatography comprises: phase A: an aqueous solution containing 0.1-0.2% (v / v) formic acid; phase B: acetonitrile.

[0089] Preferably, the mobile phase composition of the HPLC is: Phase A: 0.1% (v / v) formic acid in water; Phase B: acetonitrile.

[0090] As a preferred technical solution of an embodiment of the present invention, other conditions of high performance liquid chromatography are: flow rate: 0.25-0.35 mL / min, more preferably 0.30 mL / min. Column temperature: 25-30°C, preferably 25°C. Injection volume: 5-10 μL, preferably 5-6 μL, more preferably 6 μL; analysis time: 3.0-4.0 minutes, more preferably 3.5 minutes.

[0091] As a preferred technical solution of the embodiment of the present invention, the gradient elution conditions of high performance liquid chromatography are:

[0092] 0~1.00 minutes, phase A: 5~90%; phase B: 10~95%;

[0093] 1.01~1.50 minutes, phase A: 5~15%; phase B: 85~95%;

[0094] 1.51~2.00 minutes, phase A: 5~90%; phase B: 10~95%;

[0095] 2.01~3.50 minutes, phase A: 85~95%; phase B: 5~15%.

[0096] Preferably: 0-1.00 minutes, phase A: 10-90%; phase B: 10-90%;

[0097] 1.01-1.50 minutes, phase A: 7-10%; phase B: 90-93%;

[0098] 1.51~2.00 minutes, phase A: 10~90%; phase B: 10~90%;

[0099] 2.01~3.50 minutes, phase A: 90~93%; phase B: 7~10%.

[0100] As a preferred technical approach of the embodiment of the present invention, the gradient elution conditions of the high performance liquid chromatography are shown in Table 3:

[0101] Table 3 Gradient elution conditions of HPLC

[0102]

[0103]

[0104] As a preferred technical solution of an embodiment of the present invention, the conditions for mass spectrometry detection are:

[0105] Capillary temperature: 270-320°C; electrospray voltage: 3000-3500V; vaporization temperature: 100°C;

[0106] Sheath gas flow rate: 9-12 L / min; auxiliary gas flow rate: 1.5-4.5 L / min; lens voltage: 55-75 V; collision energy: 10-30 V.

[0107] In a specific embodiment, the mass spectrometer uses a Thermo Fisher TSQ QuantumAccess Max detector, an electrospray ion source (ESI), a positive ion mode, and selected reaction monitoring (SRM). The specific parameters are shown in Tables 4 and 5:

[0108] Table 4 Ion source parameters

[0109]

[0110] Table 5 Ion pair parameters

[0111]

[0112] The embodiment of the present invention also relates to the application of the above method in monitoring the blood concentration of polymyxin B1, B2, E1, E2 and caspofungin, wherein the test sample can be whole blood. The method of the embodiment of the present invention has a wide linear range and high sensitivity, and can accurately monitor the concentration of polymyxin B1, B2, E1, E2 and caspofungin in the blood, and can draw a blood concentration curve for studying the metabolism of related drugs.

[0113] The technical scheme of the embodiment of the present invention is further explained and illustrated by specific examples below. The raw materials of the present invention are all commercially available. The main raw materials and instruments used are as follows:

[0114] 1. Instruments and equipment:

[0115] (1) LC-MS: UltiMate3000 ultra-high performance dual ternary gradient liquid chromatograph + TSQ QuantumAccess Max triple quadrupole mass spectrometer, Thermo Scientific, USA;

[0116] (2) Centrifuge: TG18-WS high-speed centrifuge, China;

[0117] (3) Vortex mixer: MX-S, SCILOGEX, USA;

[0118] (4) Electronic analytical balance: XSE205, Mettler Toledo, USA

[0119] 2. Reagents and raw materials are shown in Table 6:

[0120] Table 6 Reagents and raw materials

[0121]

[0122] 3. Other reagents: Ultrapure water: homemade;

[0123] Acetonitrile: 4L, HPLC Grade, Merck, USA; Formic acid: 500mL, HPLC Grade, MREDA, USA; Water: drinking purified water, Wahaha, China; Porcine blank plasma: 100ml, Yuanye Biotechnology, China;

[0124] In the embodiment of the present invention, the concentrations of organic solvents such as acetonitrile and formic acid are all volume percentage concentrations.

[0125] Example 1

[0126] This embodiment proposes a method for simultaneously detecting polymyxin B1, B2, E1, E2 and caspofungin in blood by high performance liquid chromatography tandem mass spectrometry, comprising the following steps:

[0127] (I) Preparation of solution:

[0128] 1. The specification of polymyxin B sulfate reference substance is 0.50 mg. Add 1 mL of 0.2% formic acid water to dissolve it completely. The content of polymyxin B1 is 82.0%, and the content of polymyxin B2 is 9.0%. The concentration of polymyxin B1 is 410.0 mg·L -1 , polymyxin B2 concentration is 45.0 mg·L -1 of mother liquor A.

[0129] The specification of colistin sulfate reference substance is 0.50 mg. It is directly dissolved in 1 mL of 0.2% formic acid water. The content of polymyxin E1 is 30.46%, the content of polymyxin E2 is 54.64%, and the concentration of polymyxin B1 is 152.3 mg·L -1 , polymyxin B2 concentration was 273.20 mg·L -1 of mother liquor B.

[0130] The specification of caspofungin acetate reference substance is 0.50 mg. It is directly dissolved in 1 mL of 0.2% formic acid water. The purity is 98%, of which the acetate content is 5.10% and the caspofungin content is 92.90%. The concentration is 464.5 mg·L -1 of mother liquor C.

[0131] The mother solutions A, B, and C were diluted with 0.2% formic acid water to a concentration of 250 mg·L for polymyxin B1, polymyxin E2, and caspofungin. -1 , and then mixed in a volume ratio of 2:1:2. The concentration of polymyxin B1 reference substance in the obtained standard working solution was 100.00 mg·L -1 , the concentration of polymyxin B2 reference substance is 8.78 mg·L-1 , the concentration of polymyxin E1 reference substance is 27.88 mg·L -1 The concentration of polymyxin E2 reference substance is 50.00 mg·L -1 , Caspofungin reference substance concentration 100.00 mg·L -1 ;

[0132] Then continue to dilute with the above 0.2% formic acid water to prepare polymyxin B1, B2, E1, E2 and caspofungin standard working solutions with concentrations shown in Table 1;

[0133] 2. Preparation of drug-containing plasma standard solution: The seven gradient concentrations of polymyxin B1, B2, E1, E2 and caspofungin obtained by the above dilution were mixed with blank plasma at a volume ratio of 10:90 to obtain drug-containing plasma standard solution. Specifically, the concentrations of drug-containing plasma standard solutions of polymyxin B1, B2, E1, E2 and caspofungin are shown in Table 2, respectively, and stored at -20°C;

[0134] 3. Preparation of internal standard working solution: The concentration of bacitracin zinc reference stock solution is 100 mg·L -1 , diluted with 0.2% formic acid water, the concentration of bacitracin zinc in the internal standard working solution was 8 mg·L -1 The internal standard working solution was stored at -20°C.

[0135] (II) Calibration of standard solution

[0136] First, use a pipette to transfer 100 μL of drug-containing plasma standard solution, 10 μL of internal standard working solution and 200 μL of acetonitrile into 1.5 mL centrifuge tubes respectively, vortex mix at 1500 rpm for 0.5 min, centrifuge at 13000 rpm for 3 min, take 100 μL of the supernatant and dilute it with 200 μL of 0.2% formic acid water at a ratio of 1:2, take 100 μL of the diluted solution, and inject 6 μL. The diluted solution was detected by high performance liquid chromatography-mass spectrometry to obtain chromatograms of standard solutions of polymyxin B1, B2, E1, E2, caspofungin and the internal standard at seven different concentrations. The peak areas of polymyxin B1, B2, E1, E2, caspofungin and the internal standard were obtained from the chromatograms of the standard solutions of polymyxin B1, B2, E1, E2, caspofungin and the internal standard, and the peak areas of the polymyxin B1, B2, E1, E2, caspofungin and the internal standard at seven different concentrations were compared with the peak areas of the internal standard. The ratio is used as the ordinate y1 of the standard curve equation, and the ratio of the concentration in the above-mentioned polymyxin B1, B2, E1, E2, and caspofungin standard solutions to the concentration of the internal standard working solution is used as the abscissa x1 of the standard curve equation. The data of the seven different concentrations obtained by the above detection are subjected to linear regression, and the standard curve equation is fitted to be y=a*x+b, and the linear equation coefficients a and b are obtained; the standard solution is a plasma standard solution containing polymyxin B1, B2, E1, E2, and caspofungin, and the internal standard working solution is a solution containing bacitracin zinc.

[0137] (III) Sample processing

[0138] Use a pipette to transfer 100 μL of plasma sample to be tested into a 1.5 mL centrifuge tube, add 10 μL of internal standard working solution and 200 μL of acetonitrile, vortex mix at 1500 rpm for 0.5 min, centrifuge at 13000 rpm for 3 min, take 100 μL of supernatant and dilute it with 200 μL of 0.2% formic acid water at a ratio of 1:2, take 100 μL of the diluted solution, and inject 6 μL.

[0139] (IV) Testing of samples to be tested

[0140] The sample to be tested in the above step (iii) is tested using a high performance liquid chromatography-mass spectrometer to obtain a chromatogram of polymyxin B1, B2, E1, E2, caspofungin and the internal standard of the sample to be tested. The peak areas of polymyxin B1, B2, E1, E2, caspofungin and the internal standard can be obtained from the chromatogram of polymyxin B1, B2, E1, E2, caspofungin and the internal standard. The ratio y1 of the peak areas of polymyxin B1, B2, E1, E2, caspofungin and the internal standard is substituted into the standard curve equation y=a*x+b in the above step (ii). The relative concentration x of polymyxin B1, B2, E1, E2, caspofungin and the internal standard in the sample to be tested is obtained by calculation. The concentration of the internal standard working solution is known, and the concentrations of polymyxin B1, B2, E1, E2 and caspofungin in the blood sample to be tested are thus calculated.

[0141] The chromatographic column used for chromatographic analysis was Hypersil GOLD C18 2.1×100 mm, 3.5 μm; the chromatographic conditions were the same as in Table 3. The mass spectrometer was a Thermo Fisher TSQ QuantumAccess Max detector, using an electrospray ion source (ESI), positive ion detection, and selected reaction monitoring mode (SRM). The specific parameters are shown in Tables 4 and 5.

[0142] Example 2

[0143] This example is used to illustrate the linearity, matrix effect, accuracy, extraction recovery and precision of the detection method of the present invention:

[0144] 1. Linear

[0145] 100 μL of each concentration of the plasma standard solution containing polymyxin B1, B2, E1, E2, and caspofungin prepared in Example 1 were added with 10 μL of internal standard working solution, 200 μL of acetonitrile, placed in a 1.5 mL centrifuge tube, vortexed at 1500 rpm for 0.5 minutes, centrifuged at 13000 rpm for 3 minutes, 100 μL of the supernatant was taken and diluted with 200 μL of 0.2% formic acid water at a ratio of 1:2, 100 μL of the diluted solution was taken, and 6 μL of the injection volume was used for LC-MS / MS analysis. The concentrations of polymyxin B1, B2, E1, E2, and caspofungin were 0.156-10.00, 0.014-0.878, 0.044-2.788, 0.078-5.00, and 0.156-10.00 mg·L -1Within the range, according to the determination conditions of Example 1, the concentrations were determined from low to high, and the ratio of the chromatographic peak area of ​​polymyxin B1, B2, E1, E2, and caspofungin to the chromatographic peak area of ​​the internal standard - the ratio of the concentration of polymyxin B1, B2, E1, E2, and caspofungin to the concentration of the internal standard was plotted to obtain a standard curve; the results showed that the concentrations of polymyxin B1, B2, E1, E2, and caspofungin were 0.156-10.00, 0.014-0.878, 0.044-2.788, 0.078-5.00, and 0.156-10.00 mg·L -1 The linearity is good within the range, and the correlation coefficient R2﹥0.9900.

[0146] 2. Lower limit of quantification

[0147] Polymyxin B1: 0.156 mg·L -1 ; Polymyxin B2: 0.014 mg·L -1 ; Polymyxin E1: 0.044 mg·L -1 ; Polymyxin E2: 0.078 mg·L -1 ; Caspofungin: 0.156 mg·L -1 .

[0148] Matrix Effects

[0149] Polymyxin B1, B2, E1, E2, and caspofungin standard working solutions were prepared into low, medium, and high concentrations of drug-containing plasma standard solutions (each concentration value is shown in Table 7), and the matrix effect experiment was carried out. The determination was carried out according to the method of Example 1, and 6 batches were analyzed repeatedly. The results were expressed in the form of "mean ± standard deviation" of matrix effect, and the matrix effect values ​​were all within the range of 80-120% specified in the Chinese Pharmacopoeia. The matrix effect results of polymyxin B1, B2, E1, E2, and caspofungin are shown in Table 8.

[0150] Table 7 Concentration values ​​of low, medium and high drug-containing plasma standard solutions of polymyxin B1, B2, E1, E2 and caspofungin

[0151]

[0152] Table 8 Matrix effect results of polymyxin B1, B2, E1, E2, and caspofungin (n=6)

[0153]

[0154] 4. Accuracy

[0155] Polymyxin B1, B2, E1, E2, and caspofungin standard working solutions were prepared into three concentrations of drug-containing plasma standard solutions in Table 7, respectively, and the accuracy experiment was carried out. The determination was carried out according to the method of Example 1, and 6 batches were analyzed repeatedly for 3 consecutive days. The accuracy of polymyxin B1, B2, E1, E2, and caspofungin was shown in Table 9. The average accuracy of polymyxin B1, B2, E1, and E2 in the three addition levels of low, medium, and high was 97.29% to 103.80%, and the average accuracy of caspofungin in the three addition levels of low, medium, and high was 94.23% to 99.77%.

[0156] Table 9 Accuracy of polymyxin B1, B2, E1, E2, and caspofungin (n=18)

[0157]

[0158]

[0159] 5. Extraction recovery and precision

[0160] The standard working solutions of polymyxin B1, B2, E1, E2 and caspofungin were prepared into three concentrations of drug-containing plasma standard solutions in Table 7, respectively, and the extraction recovery and precision experiments were carried out. The determination was carried out according to the method of Example 1, and 6 batches were analyzed repeatedly. The extraction recovery and precision of polymyxin B1, B2, E1, E2 and caspofungin were shown in Table 10. The average recovery of polymyxin B1, B2, E1 and E2 in the range of three addition levels of low, medium and high was 38.01% to 45.36%, and the relative standard deviation RSD was 3.46% to 10.18%. The average recovery of caspofungin in the range of three addition levels of low, medium and high was 78.32% to 82.73%, and the relative standard deviation was 3.47% to 5.22%.

[0161] Table 10 Extraction recovery and precision of polymyxin B1, B2, E1, E2, and caspofungin (n=6)

[0162]

[0163] Experimental results: The chromatograms of polymyxin B1, B2, E1, E2, caspofungin, and bacitracin zinc in the standard solution are shown in Figure 1-6 The retention times of polymyxin B1, B2, E1, E2, caspofungin, and bacitracin zinc were 1.95 min, 1.93 min, 1.94 min, 1.92 min, 2.08 min, and 2.02 min, respectively.

[0164] Chromatograms of polymyxin B1, B2, E1, E2, caspofungin, and bacitracin zinc in spiked plasma samples are shown in Figure 7-12 The retention times of polymyxin B1, B2, E1, E2, and caspofungin were consistent with those of the standard solution.

[0165] Fig.13 is the chromatogram of polymyxin B1, B2, E1, E2, caspofungin and internal standard in standard solution; Fig.14 Chromatograms of polymyxin B1, B2, E1, E2, caspofungin, and internal standard in spiked plasma samples.

[0166] Based on the above validation tests, the accuracy, precision and other technical indicators of this embodiment meet the requirements (based on the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the 2020 edition of the "Chinese Pharmacopoeia"). This method detects polymyxin B1, B2, E1, E2, and caspofungin in blood with good reproducibility, small matrix effect, and stable extraction recovery, which improves the accuracy of the test results; plasma is directly sampled after protein precipitation, which makes the detection process simple and fast, reduces experimental costs, shortens analysis time, and is more conducive to high-throughput sample detection.

[0167] Example 3

[0168] The test was carried out according to the conditions of Example 1, except that: in (iii), the volume ratios of the drug-containing plasma standard solution, the internal standard working solution and acetonitrile were 100:10:200, 100:10:250, 100:10:300, 100:10:350 and 100:10:400, respectively. Then, the recovery rate was detected according to the same method as in Example 1, wherein the spiked amounts of polymyxin B1, B2, E1, E2 and caspofungin were the medium concentrations shown in Table 7. The experimental results are shown in Table 11:

[0169] Table 11 Recovery of polymyxin B1, B2, E1, E2 and caspofungin in different precipitant ratios in Example 4 (n=6)

[0170]

[0171] According to the test results in Table 10, the volume ratio of the drug-containing plasma standard solution, the internal standard working solution and acetonitrile is 100:10:200-300, which can ensure that the average fluctuation of the recovery rate of the five components does not exceed 10%.

[0172] Example 4

[0173] The test was carried out according to the conditions of Example 1, except that: in (iii), the protein precipitant was selected from the substances shown in Table 11, and then the recovery rate was detected according to the same method as in Example 1, wherein the spiked amounts of polymyxin B1, B2, E1, E2, and caspofungin were the medium concentrations shown in Table 7. The experimental results are shown in Table 12:

[0174] Table 12 Recovery of polymyxin B1, B2, E1, E2 and caspofungin in different precipitants in Example 5 (n=6)

[0175]

[0176] Among them, the concentrations of methanol and acetonitrile are volume percentage concentrations, and the concentration of 5% TCA (trichloroacetic acid) is mass percentage concentration; according to the test results in Table 12, it can be seen that the recovery rate effect of the embodiment of the present invention cannot be achieved by using other protein precipitants.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting the content of polymyxin and caspofungin, characterized in that: The steps include: S1. preparing a standard curve equation, comprising: preparing a polymyxin reference substance and a caspofungin reference substance into a standard working solution, and using bacitracin zinc as an internal standard to prepare an internal standard working solution; mixing the standard working solution and plasma to obtain a drug-containing plasma standard solution, adding the internal standard working solution and a protein precipitant to the drug-containing plasma standard solution, mixing and centrifuging, taking the supernatant and diluting it to obtain a standard solution to be tested, and detecting the standard solution to be tested by a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation, wherein the protein precipitant is acetonitrile; S2, processing to obtain a sample to be tested, including: taking the sample to be tested, adding the internal standard working solution and the protein precipitant, mixing and centrifuging, taking the supernatant and diluting to obtain the sample to be tested; S3. Testing the sample to be tested, comprising: using a high performance liquid chromatography-mass spectrometer to test the sample to be tested, substituting the test result into the standard curve equation and calculating to obtain the concentrations of polymyxin and caspofungin in the sample to be tested.

2. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S1, the polymyxins include polymyxin B1, B2, E1 and E2, the solvent of the standard working solution includes a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the concentration of the polymyxin B1 reference substance is 1.56 mg·L -1 ~100.00mg·L -1 The concentration of polymyxin B2 reference substance is 0.14 mg·L -1 ~8.78mg·L -1 The concentration of polymyxin E1 reference substance was 0.44 mg·L -1 ~27.88mg·L -1 The concentration of polymyxin E2 reference substance is 0.78 mg·L -1 ~50.00mg·L -1 The concentration of caspofungin reference substance was 1.56 mg·L -1 ~100.00mg·L -1 The solvent of the internal standard working solution includes a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the concentration of bacitracin zinc in the internal standard working solution is 5-10 mg·L -1 .

3. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S1, the volume ratio of the standard working solution and the plasma is 5-10:90-95; the conditions for mixing the standard working solution and the plasma include: vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 min, and the plasma is mammalian plasma.

4. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S1, the volume ratio of the drug-containing plasma standard solution, the internal standard working solution and the protein precipitant is 80~120:5~10:200~300, and the mixing conditions of the drug-containing plasma standard solution, the internal standard working solution and the protein precipitant include: vortex mixing at a rotation speed of 1200~1800rpm for 0.5~1 min; the centrifugation conditions include: centrifugation at a rotation speed of 12500~13500rpm for 3~5 min; the diluent of the supernatant is a formic acid aqueous solution with a volume percentage concentration of 0.1~0.3%, and the supernatant and the diluent are mixed and diluted at a volume ratio of 1:

2.

5. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S2, the volume ratio of the sample to be tested, the internal standard working solution and the protein precipitant is 80-120:5-10:200-300.

6. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S2, the conditions for mixing the sample to be tested, the internal standard working solution and the protein precipitant include: vortex mixing at a rotation speed of 1200-1800 rpm for 0.5-1 min; the conditions for centrifugation include: centrifugation at a rotation speed of 12500-13500 rpm for 3-5 min; the diluent of the supernatant is a formic acid aqueous solution with a volume percentage concentration of 0.1-0.3%, and the supernatant and the diluent are mixed and diluted at a volume ratio of 1:

2.

7. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S1 and S3, the parameter settings of the high performance liquid chromatography mass spectrometer include: the high performance liquid chromatography uses a reverse phase chromatography column C18 column, and the particle size of the C18 column is 1.8-3.8 μm; Mobile phase: Phase A: aqueous solution containing 0.1~0.2% (v / v) formic acid; Phase B: acetonitrile; Flow rate: 0.25~0.35mL / min; column temperature: 25~30℃; injection volume: 5~10µL; analysis time: 3.0~4.0min; The elution conditions of HPLC were: 0~1.00min, phase A: 5~90%; phase B: 10~95%; 1.01~1.50min, phase A: 5~15%; phase B: 85~95%; 1.51~2.00min, phase A: 5~90%; phase B: 10~95%; 2.01~3.50min, phase A: 85~95%; phase B: 5~15%.

8. The method for simultaneously detecting the content of polymyxin and caspofungin according to claim 1, characterized in that: In S1 and S3, the parameter settings of the high performance liquid chromatography-mass spectrometry instrument include: the detection conditions of the mass spectrometry include: capillary temperature: 270~320℃; electrospray voltage: 3000~3500V; vaporization temperature 100~150℃; sheath gas flow rate: 9~12L / min; auxiliary gas flow rate: 1.5~4.5L / min; lens voltage: 55~75V; collision energy: 10~30V.

9. Use of bacitracin zinc as an internal standard in the simultaneous detection of polymyxin and caspofungin content, characterized in that: The method for simultaneously detecting the content of polymyxin B and caspofungin comprises the method according to any one of claims 1 to 8.

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