阅读说明：本技术 一种检测血清中多粘菌素b1和多粘菌素b2浓度的试剂盒 (Kit for detecting concentrations of polymyxin B1and polymyxin B2 in serum ) 是由 成晓亮 李美娟 于 2020-07-14 设计创作，主要内容包括：本发明公开了一种检测血清中多粘菌素B1和多粘菌素B2浓度的试剂盒,属于药物检测技术领域。所述试剂盒包括：洗脱液、校准品溶液、内标溶液、蛋白质沉淀剂和质控品。采用本发明的试剂盒检测血清中多粘菌素B1和多粘菌素B2药物浓度,灵敏度高、特异性强、准确且前处理过程简单,5.0分钟之内完成血清中多粘菌素B1和多粘菌素B2的分离和检测,准确度与精密度基本满足要求,可用于临床上血清中多粘菌素B1和多粘菌素B2的定量分析,为临床上多粘菌素B1和多粘菌素B2药物浓度监测提供简单快速的检测方法。(The invention discloses a kit for detecting concentrations of polymyxin B1and polymyxin B2 in serum, and belongs to the technical field of drug detection. The kit comprises: eluent, calibrator solution, internal standard solution, protein precipitant and quality control substance. The kit is used for detecting the concentrations of the polymyxin B1and polymyxin B2 in serum, has high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of polymyxin B1and polymyxin B2 in serum within 5.0 minutes, basically meets the requirements on accuracy and precision, can be used for clinical quantitative analysis of polymyxin B1and polymyxin B2 in serum, and provides a simple and rapid detection method for clinical monitoring of the concentrations of the polymyxin B1and polymyxin B2 drugs.)

1. A kit for detecting the concentration of polymyxin B1and polymyxin B2 drugs in serum is characterized in that: the kit comprises: eluent, calibrator solution, internal standard solution, protein precipitator and quality control product;

the eluent consists of an eluent A and an eluent B, wherein the eluent A is 0.01-0.2% formic acid aqueous solution, and the eluent B is acetonitrile;

the calibrator solution comprises a series of mixed solutions of polymyxin B1and polymyxin B2 prepared from a blank serum matrix with known concentrations;

the internal standard solution is methanol aqueous solution of polymyxin E40000 ng/mL;

the protein precipitator is a mixed solution of methanol containing formic acid or acetic acid and acetonitrile;

the quality control product comprises mixed solution of polymyxin B1and polymyxin B2 which is prepared by blank serum matrix and has known concentration.

2. The kit of claim 1, wherein: the eluent A is 0.1% formic acid aqueous solution.

3. The kit of claim 1, wherein: the standard comprises mixed solutions of polymyxin B1and polymyxin B2 at 7 concentrations as follows:

mixing liquid I: the concentrations of polymyxin B1and polymyxin B2 are both 40 ng/mL;

and (2) mixing liquid II: the concentrations of polymyxin B1and polymyxin B2 are both 100 ng/mL;

and (3) mixing liquid III: the concentrations of polymyxin B1and polymyxin B2 are both 200 ng/mL;

and (4) mixing liquid IV: the concentrations of polymyxin B1and polymyxin B2 are both 1000 ng/mL;

and (5) mixing liquid: the concentrations of polymyxin B1and polymyxin B2 are both 2000 ng/mL;

and (6) mixing liquid six: the concentrations of polymyxin B1and polymyxin B2 are both 10000 ng/mL;

and (5) mixed liquid seven: both polymyxin B1and polymyxin B2 concentrations were 20000 ng/mL.

4. The kit of claim 1, wherein: the internal standard solution is prepared by 50% methanol aqueous solution.

5. The kit of claim 1, wherein: the volume ratio of methanol containing formic acid or acetic acid to acetonitrile in the protein precipitator is 1-3: 1-4, and the content of formic acid or acetic acid in the methanol containing formic acid or acetic acid is 0.01-0.5%.

6. The kit of claim 5, wherein: the volume ratio of methanol containing formic acid or acetic acid to acetonitrile in the protein precipitator is 2:1, and the content of formic acid or acetic acid in the methanol containing formic acid or acetic acid is 0.1%.

7. The kit of claim 1, wherein: the quality control product comprises three mixed solutions with high, medium and low concentrations:

low concentration quality control product: the concentrations of polymyxin B1and polymyxin B2 are both 80 ng/mL;

medium concentration quality control: the concentrations of polymyxin B1and polymyxin B2 are both 800 ng/mL;

high concentration quality control product: polymyxin B1and polymyxin B2 were both at 8000 ng/mL.

8. The use of the kit of claim 1 for detecting the concentrations of polymyxin B1and polymyxin B2 drugs in serum by using ultra performance liquid chromatography tandem mass spectrometry.

9. Use according to claim 8, characterized in that: the internal standard solution and the protein precipitant in the kit are mixed to prepare the protein precipitant containing the internal standard for detection, and the volume ratio of the internal standard solution to the protein precipitant is 0.1-0.3: 19.9-19.7.

10. Use according to claim 9, characterized in that: the volume ratio of the internal standard solution to the protein precipitant was 0.2: 19.8.

Technical Field

The invention belongs to the technical field of drug detection, and particularly relates to a kit for detecting drug concentrations of polymyxin B1and polymyxin B2 in serum.

Background

Polymyxin B (polymyxin B) is a lipopeptide antibiotic, wherein polymyxin B1(polymyxin B1, PMB1) and polymyxin B2(polymyxin B2, PMB2) are two main components of the polypeptide, and the polypeptide has an inhibiting effect on various negative bacteria such as pseudomonas aeruginosa, escherichia coli, klebsiella, haemophilus and the like. The product is also sensitive to other antibiotic resistant strains. But due to the serious nephrotoxicity and nerve blocking effect, the traditional Chinese medicine is only used for short-term rescue when burns and septicemia are combined or is externally used for local spray irrigation. However, the current administration schemes are mostly empirical, and a rapid and accurate method for determining polymyxin B in human body fluid is lacking, which hinders the clinical pharmacological properties of the drug. Therefore, the required concentration of the patient needs to be monitored, so that the curative effect is ensured, the toxic and side effects are reduced, and personalized administration and treatment are realized.

At present, there are also reports of methods for quantitative detection of polymyxins B1and B2, for example, the article "high performance Liquid Chromatography-Mass Spectrometry Assay for Polymyxin B1and B2 in Human Plasma" reports a method for determining the content of polymyxins B1and B2 based on a Liquid Chromatography-Mass Spectrometry combined method, but the method also has certain defects, for example, the method uses a solid phase extraction method for pretreatment, the sample dosage is large, the pretreatment is more complex and the cost is high; meanwhile, the liquid phase method mentioned in the article has gradient of 12 minutes, overlong detection time and low efficiency; moreover, the linear range detected by the method is 100-2500ng/mL, and the linear range is narrow, so that the method is not suitable for clinical analysis. For another example, the articles "Simultaneous quantification of polymyxin B1, polymyxin B2 and zymoxyxin B1-1 inhuman plasmid and linear human urine use induced colloidal phase extraction and linear chromatography-tandem mass spectrometry" report a method for determining the content of polymyxin B1and B2 based on liquid chromatography-mass spectrometry, but have certain defects, such as the method is complicated in pretreatment, requires vortex ultrasonic precipitated protein, requires detection after centrifugal concentration, and is not suitable for detection of large clinical samples, because a sample detection time is as long as 20 minutes.

Disclosure of Invention

The invention aims to provide a kit for detecting the concentrations of polymyxin B1and polymyxin B2 drugs in serum.

The invention also aims to provide application of the kit in detecting the concentrations of the polymyxin B1and polymyxin B2 drugs in serum by using an ultra performance liquid chromatography tandem mass spectrometry technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

a kit for detecting drug concentrations of polymyxin B1and polymyxin B2 in serum, comprising: eluent, calibrator solution, internal standard solution, protein precipitator and quality control product;

the eluent consists of an eluent A and an eluent B, wherein the eluent A is 0.01-0.2% formic acid aqueous solution, and the eluent B is acetonitrile;

the calibrator solution comprises a series of mixed solutions of polymyxin B1and polymyxin B2 prepared from a blank serum matrix with known concentrations;

the internal standard solution is methanol aqueous solution of polymyxin E40000 ng/mL;

the protein precipitator is a mixed solution of methanol containing formic acid or acetic acid and acetonitrile;

the quality control product comprises mixed solution of polymyxin B1and polymyxin B2 which is prepared by blank serum matrix and has known concentration.

Further, the eluent a was 0.1% formic acid aqueous solution.

Further, the standard comprises mixed solutions of polymyxin B1and polymyxin B2 in the concentration range of 7.

Further, the internal standard solution is prepared by 50% methanol aqueous solution.

Furthermore, the volume ratio of methanol containing formic acid or acetic acid to acetonitrile in the protein precipitator is 1-3: 1-4, and the content of formic acid in the methanol containing formic acid or acetic acid is 0.01-0.5%.

Further, the volume ratio of methanol containing formic acid or acetic acid to acetonitrile in the protein precipitating agent is 2:1, and the content of formic acid in the methanol containing formic acid or acetic acid is 0.1%.

Further, the quality control product comprises a mixed solution with high, medium and low concentrations.

The kit is applied to detecting the concentrations of polymyxin B1and polymyxin B2 drugs in serum by using an ultra performance liquid chromatography tandem mass spectrometry technology.

Further, the internal standard solution and the protein precipitator are mixed to prepare the protein precipitator containing the internal standard and then used for detection, and the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.

In a more preferred embodiment, the volume ratio of the internal standard solution to the protein precipitant is 0.2:19.8(1: 99).

When the kit provided by the invention is used for detecting the concentrations of polymyxin B1and polymyxin B2 in serum, the sensitivity is high, the specificity is strong, the accuracy is high, the pretreatment process is simple, the separation and detection of polymyxin B1and polymyxin B2 in serum are completed within 5.0 minutes, the accuracy and the precision basically meet the requirements, the kit can be used for clinically quantitatively analyzing polymyxin B1and polymyxin B2 in serum, and a simple and rapid detection method is provided for clinically monitoring the concentrations of polymyxin B1and polymyxin B2 drugs.

Drawings

FIG. 1 is an extracted ion flow spectrum of polymyxin B1and polymyxin B2 standards.

FIG. 2 is an extracted ion flowgram of polymyxin B1and polymyxin B2 in serum.

Detailed Description

The invention provides a kit for detecting the concentrations of polymyxin B1and polymyxin B2 drugs in serum. By using the ultra performance liquid chromatography tandem mass spectrometry technology, firstly separating an object to be detected from a serum matrix by using the ultra performance liquid chromatography, then obtaining the mass-to-charge ratio of the object to be detected and an isotope internal standard substance corresponding to the object to be detected by mass spectrometry, and calculating the contents of polymyxin B1and polymyxin B2 by using an isotope internal standard quantitative method according to an established calibration curve.

The internal standard substance corresponding to polymyxin B1and polymyxin B2 is polymyxin E.

The kit comprises the following reagents:

(1) eluent:

eluent A: 0.01 to 0.2 percent of formic acid aqueous solution; eluent B: acetonitrile;

(2) calibration solution:

a standard stock solution containing polymyxin B1(PMB1)400 μ g/mL and polymyxin B2(PMB2)400 μ g/mL was formulated in a blank serum base as a calibrator solution at seven different concentration points:

seven concentration points of polymyxin B1 are, in order: 40ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 10000ng/mL, 20000 ng/mL;

seven concentration points of polymyxin B2 are, in order: 40ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 10000ng/mL, 20000 ng/mL;

because a commercial polymyxin B2 pure standard sample cannot be obtained, polymyxin B1and polymyxin B2 are derived from a mixture polymyxin B (the ratio of polymyxin B1 is 71.1%, and the ratio of polymyxin B2 is 28.9%), polymyxin B1and polymyxin B2 are both regarded as pure standard samples, and when the concentrations of polymyxin B1and polymyxin B2 in a blood sample are calculated, the actual concentration is obtained by dividing the detected sample concentration by the respective ratio;

(3) internal standard solution:

methanol aqueous solution containing polymyxin E (PME)40000 ng/mL;

(4) protein precipitant:

a mixed solution of methanol containing formic acid or acetic acid and acetonitrile;

(5) quality control product:

blank serum matrix containing polymyxin B1and polymyxin B2 has low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,

QC (L) is the standard stock solution diluted to 5000 times with blank serum substrate,

QC (M) is the standard stock solution diluted 500 times with blank serum matrix,

qc (h) was diluted 50-fold with blank serum matrix for the standard stock solution described above.

In a preferred embodiment, the eluent a is preferably 0.1% formic acid aqueous solution.

In one scheme, the protein precipitator is a mixed solution of methanol and acetonitrile containing formic acid or acetic acid; preferably, the volume ratio of methanol to acetonitrile in the protein precipitator is 1-3: 1-4, and the content of formic acid or acetic acid is 0.01-0.5%; more preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 2:1, and the content of formic acid or acetic acid is 0.1%.

In one embodiment, the serum blank matrix is serum blank without the drug of interest.

The standard stock solutions mentioned in the present invention were prepared as follows:

the above standards were formulated at the following concentrations: polymyxin B50 mg/mL;

transferring a standard product mother solution: polymyxin B8. mu.L; then added to 992. mu.L of 50% methanol water to give 1mL of mixed standard stock solution.

The internal standard solution mentioned in the invention is prepared according to the following method:

preparing the following internal standard mother liquor by using 50% methanol water: polymyxin E20 mg/mL;

transferring internal standard mother liquor: polymyxin E2. mu.L; then adding the mixture into 998 mu L of 50% methanol water to obtain 1mL of internal standard solution; wherein, polymyxin E40000 ng/mL;

the serum mentioned in the invention is human or animal serum.

In a preferred embodiment, a kit for detecting polymyxin B1and polymyxin B2 in serum comprises the following reagents:

(1) eluent:

eluent A: 0.1% aqueous formic acid; eluent B: acetonitrile;

(2) calibration solution:

preparing the polymyxin B into a standard mother solution with the following concentration: polymyxin B50 mg/mL;

transferring a standard product mother solution: polymyxin B8. mu.L; then adding into 992. mu.L of 50% methanol water to obtain 1mL of standard stock solution; the concentrations are given in table 1 below.

TABLE 1 stock solutions of standards

The invention prepares the stock solution of the mixed standard substance into the solutions of the calibrator with seven different concentration points by using a blank serum substrate (blank serum without target drugs), and the preparation process is as follows:

adding 10 mu L of standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; diluting 50 μ L of the first high-value concentration point with 50 μ L of blank serum matrix to obtain a second high-value concentration point; diluting the first high-value concentration point with 9 times volume of blank serum substrate to obtain a third high-value concentration point; diluting the second high-value concentration point with 9 times volume of blank serum substrate to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 9 times volume of blank serum substrate to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 9 times volume of blank serum matrix to obtain a sixth high-value concentration point; and (4) taking the fifth high-value concentration point, and diluting the fifth high-value concentration point with 5 times of volume of blank serum substrate to obtain a seventh high-value concentration point.

The invention adopts a gradient dilution method to prepare standard yeast, after a standard solution is taken out from a refrigerator at the temperature of 20 ℃ below zero, the standard solution is vortexed for 10s, the highest concentration point of the standard yeast is prepared by using the standard solution within 2min, and the standard yeast is stored at the temperature of 80 ℃ below zero after the standard yeast is prepared, and the specific process is shown in the following table 2 (unit: ng/mL).

TABLE 2 Standard koji preparation

(3) Internal standard solution:

preparing the following internal standard mother liquor by using 50% methanol water: polymyxin E20 mg/mL;

transferring internal standard mother liquor: polymyxin E2. mu.L; then adding the mixture into 998 mu L of 50% methanol water to obtain 1mL of internal standard solution; the concentrations refer to table 3 below.

TABLE 3 internal standard solution preparation

(4) Protein precipitant:

the mixed solution of methanol containing formic acid and acetonitrile, wherein the volume ratio of the methanol to the acetonitrile is 2:1, and the content of the formic acid is 0.1%.

(5) Quality control product:

the mixed standard stock solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without target drugs, wherein the three different concentrations are specifically shown in table 4.

TABLE 4 corresponding concentration of quality control (unit ng/mL)

QC (L) includes: polymyxin B1(PMB1)80ng/mL and polymyxin B2(PMB2)80 ng/mL;

QC (M) comprises: polymyxin B1(PMB1)800ng/mL and polymyxin B2(PMB2)800 ng/mL;

QC (H) includes: polymyxin B1(PMB1)8000ng/mL and polymyxin B2(PMB2)8000 ng/mL.

When the kit is used for detecting polymyxin B1and polymyxin B2 in serum, the internal standard solution and the protein precipitant are mixed to prepare the protein precipitant containing the internal standard. In a preferable scheme, the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7. In a more preferred embodiment, the volume ratio of the internal standard solution to the protein precipitant is 0.2:19.8(1: 99). For example, the protein precipitant containing the internal standard is prepared by mixing an internal standard solution and a protein precipitant (methanol and acetonitrile in a volume ratio of 1:1, and 0.1% formic acid) in a volume ratio of 1: 99.

The application of the kit in detecting polymyxin B1and polymyxin B2 in serum by using an ultra performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.

The specific detection method comprises the following steps:

a method for detecting the concentration of polymyxin B1and polymyxin B2 drugs in serum comprises the steps of pretreating a serum sample, oscillating, centrifuging, taking the supernatant for sample injection, detecting polymyxin B1and polymyxin B2 in the pretreated serum by adopting an ultra high performance liquid chromatography tandem mass spectrometry technology, separating an object to be detected from a serum matrix by using the ultra high performance liquid chromatography, establishing a calibration curve by using a mass spectrum internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the polymyxin B1and polymyxin B2 drugs.

The internal standard substances corresponding to the medicines are as follows: polymyxin E.

The specific chromatographic conditions are as follows:

(1) ultra-high performance liquid chromatography conditions:

mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution; mobile phase B: acetonitrile;

the type of the chromatographic column: phenomenex Kinetex XB-C18;

a mixed mobile phase A and a mixed mobile phase B are adopted for gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 85-100: 25-0; the gradient elution procedure was as follows: in 0-1.0 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 85-100: 25-0 to 70:30 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 2:98 at a constant speed within 1.0-2.5 minutes; the volume ratio of mobile phase A and mobile phase B was maintained at 2:98 in 2.5-3.0 minutes, and changed from 2:98 to the initial ratio in 3.0-5.0 minutes, with 5.0 minutes per sample collection time.

(2) Mass spectrum conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 3.0kV (ESI +); the temperature of the drying gas is 250 ℃; the pressure of atomizing gas is 45psi, the temperature of sheath gas is 325 ℃, and the flow rate of the sheath gas is 11L/min; each target and its corresponding internal standard were monitored simultaneously.

In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid into the mobile phase A, can effectively improve the ionization efficiency of the target compound, has higher sensitivity than the detection of polymyxin B1and polymyxin B2 in serum by adopting an LC-MS/MS method in the prior art under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of polymyxin B1and polymyxin B2 within 5.0 minutes. In a preferable embodiment, the mobile phase a is 0.05% to 0.2% formic acid aqueous solution, and in the case of not affecting the effect of the present invention, the mobile phase a is preferably 0.1% formic acid aqueous solution.

In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts acetonitrile and 0.01-0.2% formic acid aqueous solution as mobile phase, the chromatographic column model is Phenomenex Kinetex XB-C18 (3.0X 50mm,2.6 μm), under the cooperation of other conditions, the endogenous substance does not interfere the determination of the sample, the sensitivity is high, the specificity is strong, and the accuracy and precision basically meet the requirements.

When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention adopts polymyxin E as an internal standard, the internal standard and an object to be detected have similar structures and the same chemical properties, and the repeatability and the accuracy are better when the polymyxin B1and the polymyxin B2 in serum are measured.

In a preferable scheme, the initial ratio of the mobile phase A to the mobile phase B is 85-100: 25-0. Further preferably, the initial ratio of mobile phase a and mobile phase B is 99: 1.

In a preferred embodiment, the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min.

Further, the column temperature is 40-60 ℃, preferably 50 ℃.

In one embodiment, the injection volume is 0.2-10 μ L, preferably 1 μ L.

In a preferred scheme, when the ultra performance liquid chromatography tandem mass spectrometry technology is adopted to detect polymyxin B1and polymyxin B2 in pretreated serum, the specific chromatographic conditions are as follows:

(1) ultra-high performance liquid chromatography conditions:

mobile phase A: 0.1% aqueous formic acid; mobile phase B: acetonitrile;

the type of the chromatographic column: phenomenex Kinetex XB-C18 (3.0X 50mm,2.6 μm);

adopting a mode of gradient elution by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the initial ratio of the mobile phase A to the mobile phase B is 99: 1; the gradient elution procedure was as follows: in 0-1.0 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 85-100: 25-0 to 70:30 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 2:98 at a constant speed within 1.0-2.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is kept at 2:98 within 2.5-3.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is changed from 2:98 to the initial ratio within 3.0-5.0 minutes, and the collection time of each sample is 5.0 minutes; the gradient elution mode is specifically shown in table 1; the flow rate was 0.3mL/min, the column temperature was 50 ℃ and the injection volume was 1. mu.L.

TABLE 5 mobile phase gradient elution parameters

(2) Mass spectrum conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 3.0kV (ESI +); the temperature of the drying gas is 250 ℃; the pressure of atomizing gas is 45psi, the temperature of sheath gas is 325 ℃, and the flow rate of the sheath gas is 11L/min; the mass spectrum source parameters are shown in table 6, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are simultaneously monitored and shown in table 7.

TABLE 6 Mass Spectrometry Source parameters

TABLE 7 determination of Mass Spectrometry parameters for polymyxin B1and polymyxin B2

The serum mentioned in the invention is human or animal serum.

In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing internal standard work into the centrifuge tube, oscillating for 3-10 min, centrifuging for 4-10 min at 12000-15000 r/min and 4-20 ℃, taking supernatant into a sample injection bottle, and performing sample injection; the protein precipitator containing the internal standard is prepared by mixing an internal standard solution and a protein precipitator, wherein the volume ratio of methanol to acetonitrile in the protein precipitator is 2:1, and the content of formic acid is 0.1%.

In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing internal standard work into the centrifuge tube, oscillating for 3-10 min, centrifuging for 4-10 min at 12000-15000 r/min and 4-20 ℃, taking supernatant into a sample injection bottle, and performing sample injection; the protein precipitator containing the internal standard is prepared by mixing an internal standard solution and a protein precipitator, wherein the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.

In a more preferred embodiment, the pretreated serum of the present invention is prepared as follows: 50 mu L of serum is taken to be put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 2:1, and 0.1% formic acid is contained) is added into the centrifuge tube, the mixture is shaken for 5min, and after centrifugation is carried out for 5min at 14000r/min and 15 ℃, 60 mu L of supernatant is taken to be put into a sample injection bottle, and 1 mu L of sample injection is carried out, wherein the protein precipitant containing internal standard is prepared by mixing internal standard solution and protein precipitant, and the volume ratio of the internal standard solution to the protein precipitant is 0.2:19.8 (namely 1: 99).

In one embodiment, the protein precipitant containing the internal standard is prepared as follows:

preparing the following internal standard mother liquor by using 50% methanol water: polymyxin E20 mg/mL;

transferring internal standard mother liquor: polymyxin E2. mu.L; adding into 998 μ L methanol water to obtain 1mL internal standard solution;

and adding 100 mu L of the internal standard solution into 19.8mL of protein precipitant to obtain the protein precipitant containing the internal standard.

In one scheme, the protein precipitator is a methanol and acetonitrile mixed solution containing formic acid; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1-3: 1, and the protein precipitant contains 0.01-0.2% of formic acid. More preferably, the protein precipitant has a methanol to acetonitrile volume ratio of 2: 1and contains 0.1% formic acid.

In one embodiment, the standard solution is prepared as follows:

preparing a standard mother solution with the following concentration: polymyxin B50 mg/mL;

polymyxin B8. mu.L; then added to 992. mu.L of 50% methanol water to give 1mL of a standard stock solution. The standard stock solution comprises: polymyxin B1(PMB1) 400. mu.g/mL, and polymyxin B2(PMB2) 400. mu.g/mL.

Preparing the standard stock solution into calibration solution with seven different concentration points by using a blank serum substrate, wherein the seven concentration points of the calibration solution are as follows:

seven concentration points of polymyxin B1 are, in order: 40ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 10000ng/mL, 20000 ng/mL;

seven concentration points of polymyxin B2 are, in order: 40ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 10000ng/mL, 20000 ng/mL.

50 mu L of each concentration point sample is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 2:1, and the formic acid is 0.1 percent) is added into the centrifuge tube, the mixture is shaken for 5min, and after centrifugation is carried out for 5min at 14000r/min and 15 ℃, 60 mu L of supernatant is taken and put into a sample injection bottle, and 1uL of sample injection is carried out.

The invention also comprises a quality control product prepared by the following method: preparing the mixed standard stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without target drugs, wherein,

qc (l) was a 5000-fold dilution of the above standard stock solution in blank serum matrix.

Qc (m) is a 500-fold dilution of the above standard stock solution in blank serum matrix.

Qc (h) was diluted 50-fold with blank serum matrix for the standard stock solution described above.

QC (L) includes: polymyxin B1(PMB1)80ng/mL and polymyxin B2(PMB2)80 ng/mL.

QC (M) comprises: polymyxin B1(PMB1)800ng/mL and polymyxin B2(PMB2)800 ng/mL.

QC (H) includes: polymyxin B1(PMB1)8000ng/mL and polymyxin B2(PMB2)8000 ng/mL.

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.