CN111830168A - LC-HR-MS/MS quantitative analysis method of poloxamer - Google Patents
LC-HR-MS/MS quantitative analysis method of poloxamer Download PDFInfo
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Abstract
The invention relates to a LC-HR-MS/MS quantitative analysis method of poloxamer, which specifically comprises the following steps: firstly, respectively preparing a liquid to be detected and a standard liquid; then carrying out liquid chromatography-mass spectrometry combined detection analysis; wherein the high performance liquid chromatography adopts PLRP-S Reversed-Phase Column chromatography for gradient elution; in elution, 0.1 to 0.3 percent aqueous solution of formic acid is used as a mobile phase A phase, and 0.1 to 0.3 percent acetonitrile and isopropanol of formic acid are used as a mobile phase B phase; the condition parameters of the mass spectrometry are as follows: ion spray voltage: 5000-6000V, ion source temperature: 450-550 ℃, collision energy: 40 to 50 eV; and calculating the content of poloxamer in the liquid to be detected according to the peak area ratio. The LC-HR-MS/MS quantitative analysis method of poloxamer has the advantages of strong selectivity, high sensitivity and short analysis time.
Description
Technical Field
The invention relates to an analysis method of a pharmaceutical excipient, in particular to an LC-HR-MS/MS quantitative analysis method of poloxamer.
Background
Poloxamers have unique physicochemical properties and good biocompatibility and are approved by the FDA as food additives, pharmaceutical excipients and drug delivery vehicles. In recent years, poloxamers have found widespread use in the pharmaceutical and biomedical fields. Despite the numerous reports on the use of poloxamers, the pharmacokinetics of these and related formulations have been poorly studied. There is no literature on whether poloxamers can be absorbed into the blood circulation after oral administration or topical administration based on poloxamer in situ gels. The absorption, distribution, metabolism and excretion processes of poloxamers of different molecular weights in vivo are not yet fully understood.
The in vitro and in vivo quantitative analysis methods of poloxamers reported in the literature so far mainly comprise a colorimetric method, a radiolabelling method, a size exclusion chromatography method and the like. (1) A colorimetric method: the method mainly uses cobalt thiocyanate or iodine-potassium iodide as a color developing agent, has low sensitivity and poor reproducibility and selectivity, and can only roughly estimate the content of poloxamer in a sample. (2) Radiolabelling: an isotope (e.g., 14C) is labeled onto the carbon skeleton of the poloxamer and the poloxamer is quantitated by measuring the amount of radioactive atoms in the sample. However, the radioactive isotope can cause pollution to the environment, is difficult to be applied to human body tests, the marking of the poloxamer must be finished in a laboratory with special protection equipment, and a clear methodology is not established to show whether the pharmacokinetic parameters of the poloxamer are changed before and after the marking. (3) Size Exclusion Chromatography (SEC): also known as gel permeation chromatography, is mainly used for separation of macromolecular substances. The method has reported detectors such as differential refractive index detector, evaporative light scattering detector and mass spectrometry detector. The differential refractive index detector can be used for in vivo content determination of poloxamer 188, the lower limit of quantification is 25 mug/mL, the sensitivity is low, the base line is unstable, and the equilibrium time is long. Measuring the content of poloxamer 188 in the itraconazole injection by an evaporation photodetector and an electrospray ionization mass spectrometry (ESI-MS), wherein the lower limit of the quantification of the poloxamer 188 in the itraconazole injection is 25 mu g/mL, and the linear range of the itraconazole injection is narrow; the latter adopts a selective ion monitoring mode, uses the strongest peak m/z 976.4 as a poloxamer 188 quantitative tracking ion, and because poloxamer has non-unique and normally distributed molecular weight and has multiple charges in an ESI ionization mode, the information of many other ions can be lost when only a certain specific ion is selected for analysis, and the whole information acquisition cannot be obtained. The analysis methods cannot accurately evaluate the quality of poloxamer, cannot avoid the interference of chemical noise with the same mass-to-charge ratio, and cannot accurately evaluate the pharmacokinetic behavior in vivo. Therefore, a method which has high sensitivity and good selectivity and can meet the requirement of in vivo poloxamer content measurement needs to be developed, and a good methodology basis is provided for the pharmacokinetic research of the poloxamer content.
Disclosure of Invention
The invention provides an LC-HR-MS/MS quantitative analysis method of poloxamer, in particular to poloxamer 188; the method mainly aims to establish an LC-HR-MS/MS quantitative analysis method which has strong selectivity, high sensitivity and short analysis time, and can provide reference for in vivo quantitative analysis of other types of poloxamers. The manuscript finalizing analysis method provided by the invention provides a good basis for the follow-up poloxamer pharmacokinetic research, and provides a certain reference for the pharmacological research and safety evaluation of the polymer.
The LC-HR-MS/MS quantitative analysis method comprises the following steps:
1) respectively preparing a sample solution to be detected and a standard sample solution; adding an internal standard working solution into the sample solution to be detected and the standard sample solution;
2) carrying out liquid chromatography-mass spectrometry detection analysis on the standard sample solution and the sample solution to be detected;
the high performance liquid chromatography adopts a PLRP-S Reversed-Phase Column chromatography, takes 0.1-0.3% formic acid water solution as a mobile Phase A Phase, takes 0.1-0.3% formic acid acetonitrile and isopropanol as a mobile Phase B Phase, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; gradient elution was performed as follows:
0-1.4 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
1.4-1.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 40% to 65%;
1.5-3.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 65%;
3.0-3.1 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 65% to 95%;
3.1-4.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is 95%;
4.5-4.6 min: the percentage of the mobile phase B in the total volume of the mobile phase is reduced to 40 percent from 90 percent;
4.6-6.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
wherein, the condition parameters of the mass spectrometry are as follows: ion spray voltage: 5000-6000V, ion source temperature: 450-550 ℃, collision energy: 40 to 50 eV;
3) and calculating the content of the poloxamer in the sample solution to be detected.
Wherein, the aperture of the Agilent PLRP-S Reversed-Phase chromatographic column provided by the high performance liquid chromatography is
The effect of adsorbing poloxamer 188 is remarkable, and the poloxamer 188 is not easily interfered.
The invention further provides that the sample in the sample solution to be detected is a solution to be detected of plasma or visceral organs; and treating the solution to be detected by adopting a protein precipitation method to obtain the sample solution to be detected. Specifically, when blood plasma is used as a liquid to be detected, a solvent is directly added for protein precipitation; if viscera is used as the liquid to be detected, firstly, the viscera is smashed by adding normal saline and prepared into homogenate (namely, the pretreatment is carried out as follows); then adding solvent to precipitate protein.
Preferably, the protein precipitation treatment specifically comprises: acetonitrile and isopropanol of 0.1-0.3% formic acid are used as precipitation reagents, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; adding a precipitation reagent into the liquid to be detected according to the volume ratio of 3-5: 1 to precipitate protein; centrifuging at high speed after swirling, and taking supernatant to obtain the sample solution to be detected;
in actual operation, the solution to be detected and the internal standard working solution can be added with a precipitation reagent at the same time; if the volume ratio of the liquid to be detected to the internal standard working solution is 1:1, the volume ratio of the sample solution to be detected to the internal standard working solution is about 1:1 after protein precipitation.
More preferably, the organ further comprises a pretreatment, specifically: stirring and crushing the in vitro viscera to homogenate, and centrifuging at high speed to obtain supernatant fluid to obtain a fluid to be detected;
the invention further provides that the standard solution is prepared by adopting the following method:
preparing at least 6 gradient poloxamer standard solutions of 2.5-250 mug/mL; the solvent of the poloxamer standard solution is acetonitrile and water in a volume ratio of 2: 2.5-3.5. In actual operation, high-concentration poloxamer standard solution can be prepared; then adding a certain proportion of blank plasma for mixing.
The invention further provides that an internal standard working solution is added into the sample solution to be detected and the standard sample solution, wherein the internal standard working solution is 0.8-1.2 mug/mL simvastatin solution, and the solvent of the simvastatin solution is acetonitrile and water in a volume ratio of 2: 2.5-3.5.
Most preferably, the internal standard working solution is a simvastatin solution with the volume ratio of 1 mug/mL, and the solvent of the simvastatin solution is acetonitrile and water with the volume ratio of 2: 3.
The volume ratio of the sample solution to be detected to the internal standard working solution is 1: 0.8-1.2, and preferably 1: 1; the volume ratio of the standard sample solution to the internal standard working solution is 1: 0.8-1.2, and preferably 1: 1.
The simvastatin is used as an internal standard, so that the accuracy and precision of quantitative analysis can be improved, and the quantitative analysis method can be more stable. Generally, the stable isotope labeling internal standard and the structural analogue internal standard can better improve the accuracy and precision of analysis, but poloxamer 188 is used as a high molecular polymer, and the stable isotope labeling internal standard is difficult to synthesize, so that a small molecular compound simvastatin is selected as the internal standard. The internal standard substance has similar retention time with poloxamer 188, generates different ion fragments for quantification, does not generate cross interference on poloxamer 188, and has good sensitivity and reproducibility.
The invention further provides that the conditions of the high performance liquid chromatography at least comprise the following items:
the column temperature is 35-45 ℃,
the flow rate is 0.7-0.9 mL/min;
the temperature of the automatic sample injector is 12-18 ℃;
preferably, the conditions of the high performance liquid chromatography further include at least one of:
taking a 0.1% formic acid aqueous solution as a mobile phase A phase, taking 0.1% formic acid acetonitrile and isopropanol as a mobile phase B phase, wherein the volume ratio of the acetonitrile to the isopropanol is 2: 3;
the column temperature was 40 c,
the flow rate is 0.8 mL/min;
autosampler temperature 15 ℃.
The mobile phase A in the high performance liquid chromatography is the mixture of formic acid, acetonitrile and isopropanol; in the experimental process, after isopropanol with stronger elution capacity is added, the residue of the analyte is reduced, and the peak width is narrowed; the effects of acetonitrile and isopropanol with different proportions are obviously different; when the ratio is acetonitrile: the best elution effect and the best peak pattern were obtained with isopropanol (2:3, v/v). In addition, 0.1% formic acid is added into the mobile phase A provided by the invention, so that the ionization efficiency and the chromatographic peak pattern can be obviously improved.
The invention further optimizes the gradient elution mode on the basis of adopting the mobile phase. During the experiment, it was found that poloxamer 188 was able to remain on the column in 40% of the organic phase, with poloxamer 188 having the highest response and the best peak pattern in 65% of the organic phase. However, since 65% of the organic phase does not completely wash away the interfering components in the biological sample, 95% of the organic phase is used again at the end of the gradient for washing away the interfering components.
In addition, flow rate, column temperature, and autosampler temperature all contribute to elution efficiency. The larger the flow rate is, the higher the column pressure of the chromatographic column is, and the peak type of the chromatographic column is also influenced to a certain extent, and finally the flow rate is determined to be 0.8 mL/min. Column efficiency is also affected by the column temperature and the autosampler temperature, and the appropriate temperature can make the analyte more sensitive.
The invention further provides that the mass spectrometry conditions further include at least one of the following:
air curtain air: 30-40 psi;
GS1, 35-55 psi; preferably 35-45 psi;
GS2, 35-55 psi; preferably 35-45 psi;
cluster-splitting voltage: 90-110V;
collision energy: 40 to 50 eV;
MSALLscanning range: 50-1250 Da;
ion extraction window width for extracting poloxamer 188 characteristic fragment ions: 0.01 Da;
preferably, the conditions of mass spectrometry are: ion spray voltage: 5500V, ion source temperature: 500 ℃, gas curtain gas: 35psi, GS1:40psi, GS2:40psi, declustering voltage: 100V, collision energy: 45eV, MSALLScanning range: 50-1250Da, and the width of an ion extraction window for extracting poloxamer 188 characteristic fragment ions: 0.01 Da.
The mass spectrometry provided by the invention adopts electrospray ionization, has high ionization efficiency, and is more suitable for analyzing high molecular polymers and strong polar compounds such as poloxamer 188.
In order to remove other interfering ions of poloxamer 188 as much as possible and ensure the quantitative lower limit signal intensity, the ion extraction window for extracting the fragment ions of poloxamer 188 features has the width of +/-0.01 Da, and the signal-to-noise ratio of the width of the extraction window is high.
According to the invention, the mass spectrum cracking rule of poloxamer 188 is explored, comparative research and feasibility analysis of poloxamer in different mass spectrum scanning modes are carried out, and research finds that a TOF MS scanning mode is an effective method for quantifying poloxamer 188.
In optimizing CE, it was found that when the energy of CE is small, the distribution of poloxamer 188 fragment ions is made up of fragment ions with relatively large mass-to-charge ratios, although selectivity may be better, these precursor ions respond very poorly, which may lead to low sensitivity of the quantitation result, and not all poloxamer 188 molecules produce these same fragments without substitutability. As the CE energy increases, the non-countable poloxamer 188 precursor ions are gradually fragmented into a limited number of singly charged ions, which are a common structure of poloxamer 188 molecules and are suitable as quantitative ions.
TOF MS scan mode can convert an infinite number of study analysis objects into a limited number of characteristic ions compared to precursor ion scan mode, making polydisperse quantitative analysis of polymers possible. And the secondary ion stability of the TOF MS mode is good, the reproducibility is good, and the anti-interference capability is strong. Compared with the in-source fragmentation mode, the TOF MS scanning mode has large CE energy, so that poloxamer 188 polymer is fully fragmented, and the response of characteristic fragment ions is greatly improved. And all poloxamer 188 ions can be broken up, and the accuracy of a given amount also provides guarantee.
The invention further provides that the step 3) is specifically as follows:
performing LC-MS/MS quantitative analysis on a standard sample solution, performing linear regression operation by using the peak area ratio of the standard sample solution to an internal standard substance and adopting a weighted least square method, wherein a weight factor W is 1/X2(ii) a Calculating by quantitative analysis software Analyst 1.7.1 to obtain the back-calculated concentration and linear regression equation of the standard curve sample; and comparing the peak area ratios of the sample solution to be detected and the internal standard substance, and calculating the content of poloxamer in the sample solution to be detected based on the linear regression equation.
The invention provides a preferable scheme, namely LC-MS/MS quantitative analysis of poloxamer 188, which is characterized by comprising the following steps:
1) preparing a sample solution to be tested: treating the solution to be detected by adopting a protein precipitation method; specifically, the method comprises the following steps: acetonitrile and isopropanol of 0.1-0.3% formic acid are used as precipitation reagents, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; adding a precipitation reagent into the liquid to be detected according to the volume ratio of 3-5: 1, and precipitating protein; centrifuging at high speed after vortexing, and taking supernate to obtain the sample solution to be detected;
wherein the liquid to be detected is blood plasma to be detected or organ to be detected after pretreatment; the pretreatment specifically comprises the following steps: crushing the in vitro viscera into homogenate, and centrifuging at high speed to obtain supernatant fluid to obtain fluid to be detected;
2) preparation of standard sample solutions: preparing at least 6 gradient poloxamer 188 standard solutions of 2.5-250 mug/mL; the solvent of the poloxamer standard solution is acetonitrile and water in a volume ratio of 2: 2.5-3.5.
Adding an internal standard working solution into the to-be-detected sample solution and the standard sample solution according to the volume ratio of 1: 0.8-1.2 respectively; the internal standard working solution is a simvastatin solution with the volume ratio of 0.8-1.2 mug/mL, and the solvent of the simvastatin solution is acetonitrile and water with the volume ratio of 2: 2.5-3.5;
3) carrying out liquid chromatography-mass spectrometry detection analysis on the standard sample solution and the sample solution to be detected;
the high performance liquid chromatography adopts a PLRP-S Reversed-Phase Column chromatography, takes 0.1-0.3% formic acid water solution as a mobile Phase A Phase, takes 0.1-0.3% formic acid acetonitrile and isopropanol as a mobile Phase B Phase, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; gradient elution was performed as follows:
0-1.4 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
1.4-1.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 40% to 65%;
1.5-3.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 65%;
3.0-3.1 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 65% to 95%;
3.1-4.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is 95%;
4.5-4.6 min: the percentage of the mobile phase B in the total volume of the mobile phase is reduced to 40 percent from 90 percent;
4.6-6.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
the column temperature is 35-45 ℃, and the flow rate is 0.7-0.9 mL/min; the temperature of the automatic sample injector is 12-18 ℃;
wherein, the condition parameters of the mass spectrometry are as follows: ion spray voltage: 5000-6000V, ion source temperature: 450-550 ℃, collision energy: 40 to 50 eV; air curtain air: 30-40 psi; GS1, 35-45 psi; GS2, 35-45 psi; cluster-splitting voltage: 90-110V; collision energy: 40 to 50 eV; MS (Mass Spectrometry)ALLScanning range: 50-1250 Da; ion extraction window width for extracting poloxamer 188 characteristic fragment ions: 0.01 Da;
4) performing LC-MS/MS quantitative analysis, performing linear regression by using the peak area ratio of the standard sample solution to the internal standard substance and adopting a weighted least square method, wherein the weight factor W is 1/X2(ii) a Calculating by quantitative analysis software Analyst 1.7.1 to obtain the back-calculated concentration and linear regression equation of the standard curve sample; and comparing the peak area ratio of the sample solution to be detected and the internal standard substance, and calculating the content of poloxamer in the sample solution to be detected based on the linear regression equation.
Drawings
FIG. 1 is a plasma chromatogram of different interfering components provided in the examples;
FIG. 2 is a linear regression graph of a standard curve obtained by the quantitative analysis method provided in the examples.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The following examples reagents and equipment are specified below:
1. reagent
TABLE 1 reagents used in connection with
2. Instrument for measuring the position of a moving object
TABLE 2 associated instruments
3. The following drugs were used:
the substance to be tested: poloxamer 188, source: beijing MREDA, molecular formula: h (OCH)2CH2)x(OCH2CHCH3)y(OCH2CH2)zOH, theoretical average molecular weight: 8400, purity: 99.9%, storage conditions: room temperature, CAS No.: 9003-11-6;
internal standard: simvastatin, source: sigma company, usa, molecular formula: c25H38O5Theoretical molecular weight: 418.57
The animals used in this experiment were as follows:
SPF grade healthy SD male rats grow for about 7 weeks, each rat weighs 200 + -20 g, and the license number is SCXK (Liao) 2015-0001.
Preparation of rat blank plasma: specifically, whole blood is collected through orbital venous plexus of healthy SD rats, and centrifuged for 5min at 13000r/min by a high-speed centrifuge, and supernatant is taken.
Examples
This example provides an LC-MS/MS quantitative analysis of poloxamers, comprising the steps of:
1) preparing an internal standard working solution: the internal standard working solution is 1 mu g/mL simvastatin solution, and the solvent of the simvastatin solution is acetonitrile and water in a volume ratio of 2: 3.
Specifically, the preparation of the internal standard stock solution (1mg/mL) is specifically as follows: weighing 25.0mg of simvastatin in an analytical balance, placing the simvastatin in a 25mL volumetric flask, adding a small amount of acetonitrile-water (2:3, v/v), shaking the volumetric flask, adding acetonitrile-water (2:3, v/v) to fix the volume to a scale mark after the simvastatin is completely dissolved, and obtaining a simvastatin stock solution with the final concentration of 1.0 mg/mL; dilute to 1. mu.g/mL with acetonitrile-water (2:3, v/v).
2) Preparation of standard sample solutions: preparing 7 gradient poloxamer 188 standard solutions of 2.5-250 mug/mL, specifically 250, 125, 75, 25, 12.5, 7.5 and 2.5 mug/mL; the solvent of the poloxamer standard solution is acetonitrile and water in a volume ratio of 2: 3.
Specifically, the method comprises the following steps: weighing 25.0mg of poloxamer 188 by an analytical balance, placing the poloxamer 188 into a 25mL volumetric flask, adding a small amount of acetonitrile-water (2:3, v/v), shaking the volumetric flask, adding the acetonitrile-water (2:3, v/v) to a constant volume to a scale mark after the poloxamer 188 is completely dissolved, and obtaining a poloxamer 188 stock solution with the final concentration of 1.0mg/mL, wherein the solution is clear and transparent; then diluted with acetonitrile-water (2:3, v/v) solution to concentrations of 250, 125, 75, 25, 12.5, 7.5, 2.5. mu.g/mL. Adding internal standard working solution into 7 gradient poloxamer 188 standard solutions according to the volume ratio of 1: 1.
3) Preparing a sample solution to be tested: treating the solution to be detected by adopting a protein precipitation method; specifically, the method comprises the following steps: taking acetonitrile of 0.1% formic acid and isopropanol as precipitation reagents, wherein the volume ratio of the acetonitrile to the isopropanol is 2: 3; adding the liquid to be detected and the internal standard working solution into the solvent according to the volume ratio of 1:1, and precipitating protein; then carrying out high-speed centrifugation, and taking supernatant to obtain the sample solution to be detected; wherein the volume of the precipitation reagent and the liquid to be detected is 4: 1.
Wherein the liquid to be detected is blood plasma to be detected or organ to be detected after pretreatment; the pretreatment specifically comprises the following steps: crushing the in vitro viscera into homogenate, and centrifuging at high speed to obtain supernatant fluid to obtain fluid to be detected;
specifically, the method comprises the following steps: the specific preparation method of the sample solution of the blood plasma to be detected comprises the following steps: precipitation reagent was 0.1% formic acid-acetonitrile: isopropanol (2:3, v/v). To a 2mL anti-adsorption EP tube were added 50 μ L of internal standard working solution (1 μ g/mL) and 50 μ L of unknown sample of poloxamer 188 in sequence, followed by 200 μ L of 0.1% formic acid-acetonitrile: precipitation of the protein was carried out in isopropanol (2:3, v/v). Placing the mixed system in a high-speed centrifuge after the mixed system is swirled for 30s, centrifuging for 5min at the rotating speed of 13000r/min, and taking supernatant;
specifically, the method comprises the following steps: a total of 8 tissues of the mouse heart, liver, spleen, lung, kidney, stomach, muscle and brain were collected. After the tissue is taken out, the surface blood and residues in the stomach are simply washed by normal saline, the surface water is absorbed by filter paper, and the tissue is cut into pieces. 1.0. + -. 0.1g of tissue was taken out, and physiological saline (1:4, g/mL) was added thereto in an amount of 4 times the weight of the taken-out tissue, and the mixture was kneaded by a tissue homogenizer to prepare a homogenate. About 1mL of the homogenate was removed and the unpulped tissue was sunk to the bottom of the EP tube at 6000 rpm. Take 50 μ L of the homogenate supernatant into a 2mL anti-adsorption EP tube, then add 50 μ L of internal standard working solution (1 μ g/mL), then add 200 μ L of 0.1% formic acid-acetonitrile: isopropanol (2:3, v/v) was used for sample treatment. Placing the mixed system in a high-speed centrifuge after swirling for 30s, centrifuging for 5min at the rotating speed of 13000r/min, pouring the supernatant into a 2mL anti-adsorption EP tube, and taking the supernatant;
4) carrying out liquid chromatography-mass spectrometry detection analysis on the standard sample solution and the sample solution to be detected;
wherein the high performance liquid chromatography adopts a PLRP-S Reversed-Phase Column chromatography, takes 0.1% formic acid water solution as a mobile Phase A Phase, takes 0.1% formic acid acetonitrile and isopropanol as a mobile Phase B Phase, and the volume ratio of the acetonitrile to the isopropanol is 2: 3; gradient elution was performed as follows:
0-1.4 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
1.4-1.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 40% to 65%;
1.5-3.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 65%;
3.0-3.1 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 65% to 95%;
3.1-4.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is 95%;
4.5-4.6 min: the percentage of the mobile phase B in the total volume of the mobile phase is reduced to 40 percent from 90 percent;
4.6-6.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
the column temperature is 40 ℃, and the flow rate is 0.8 mL/min; the autosampler temperature was 15 ℃;
wherein, the preparation of the mobile phase is specifically as follows: 200mL of acetonitrile and 300mL of isopropanol are measured by using a measuring cylinder, poured into a mobile phase glass bottle, added with 500 mu L of formic acid, shaken gently and mixed uniformly, and subjected to ultrasonic treatment for 15min to prepare a mixture containing 0.1% formic acid-acetonitrile: isopropanol (2:3, v/v) solution; the preparation process of the mobile phase water phase comprises the following steps: accurately measuring 500mL of pure water by using a measuring cylinder, pouring the pure water into a glass bottle, adding 500 mu L of formic acid, shaking gently and mixing uniformly, and carrying out ultrasonic treatment for 15min to prepare a 0.1% formic acid-water solution.
The condition parameters of the mass spectrometry are as follows: ion spray voltage: 5500V, ion source temperature: 500 ℃, gas curtain gas: 35psi, GS1:40psi, GS2:40psi, declustering voltage: 100V, collision energy: 45eV, MSALL scan range: 50-1250Da, ion extraction window width for extracting poloxamer 188 characteristic fragment ions: 0.01 Da.
5) Performing LC-MS/MS quantitative analysis, performing linear regression by using the peak area ratio of the standard sample solution to the internal standard substance and adopting a weighted least square method, wherein the weight factor W is 1/X2(ii) a Calculating by quantitative analysis software Analyst 1.7.1 to obtain the back-calculated concentration and linear regression equation of the standard curve sample;
preparing 7 concentration levels of poloxamer 188 standard sample solutions according to the preparation method, and carrying out three ordinary experiments; one set of standard curve samples was injected at the beginning of an analysis batch, one set at the middle and one set at the end.
Performing linear regression operation by using weighted least square method, wherein the weight factor W is 1/X2The relative error of the measured value of each concentration point is reduced to the minimum, and the accuracy of the low-concentration sample is ensured to be the best. And (3) obtaining the back-calculated concentration and the linear back-return equation of the standard curve sample by utilizing the ratio of the peak area of the poloxamer 188 to be detected to the peak area of the internal standard through the calculation of quantitative analysis software Analyst 1.7.1. The concentration calculated back from the standard curve should generally be within + -15% of the indicated value,LLOQ (lower limit of quantitation) should be within ± 20% and contain a minimum of 6 effective concentrations. Linear correlation coefficient r of standard curve2Should not be less than 0.98, r2The closer to 1, the better the linear relationship is illustrated. The experiment is carried out with r2Not less than 0.99 as the standard, can meet the needs of quantitative analysis in vivo;
and comparing the peak area ratio of the sample solution to be detected and the internal standard working solution, and calculating the content of poloxamer in the sample solution to be detected based on the linear regression equation.
This experiment was performed by MS using Analyst 1.7.1 data processing software (Sciex, USA)ALLFull scan mode (TOF MS scan mode), scanning all ions of mass to charge ratio within a set range, obtaining a total ion flow map (TIC). Inputting the mass-to-charge ratio range of the needed poloxamer 188 ions, and extracting quantitative ion information from the TIC diagram to obtain the extracted quantitative chromatogram (EIC). The quality extraction window can also be optimized using Peakview 2.2 software. The quantitative ions in the test are fragments formed by two polyoxypropylene (PPO) units, the theoretical mass-to-charge ratio of the fragments is 117.0899, and the mass extraction window is 117.08-117.10.
Examples of the experiments
The LC-MS/MS quantitative analysis method provided in the above example can be used for verifying the reliability of the quantitative analysis method for poloxamer 188 in several aspects, including selectivity, standard curve, lower limit of quantitation, accuracy and precision, matrix effect, extraction recovery, residue investigation and necessary stability investigation.
1. Selectivity is
Selectivity (Selectivity) was assessed by analyzing a blank bio-matrix sample without the internal standard of poloxamer 188 test substance in comparison to the lower limit of quantitation. 6 rat blank plasma samples from different sources were randomly selected and processed in the same manner as in the above example. The response of the interfering component should be, as required, less than 20% of the analyte quantitation lower limit response and less than 5% of the internal standard response. As shown in fig. 1 and table 3, a in fig. 1 is a white blood chromatogram of rat; b is the chromatogram of rat blank plasma added with internal standard (1 mug/mL); c is LLOQ (quantitative lower limit) chromatogram (in the figure, I is poloxamer 188, RT (poloxamer 188) is 2.48 min, II is internal standard, RT (internal standard) is 2.69min), and no obvious chromatographic peak interference exists at the peak positions of the object to be detected and the internal standard, which indicates that the interference component in the plasma does not influence the quantitative analysis of poloxamer 188.
TABLE 3 examination of Poloxamer 188 Selectivity
2. Standard curve
Poloxamer 188 standard sample solutions were prepared at 7 concentration levels according to the above examples, three experiments in parallel. LC-MS/MS quantitative analysis was performed, with one set of standard curve samples injected at the beginning of an analysis batch, one set at the middle and one set at the end. Performing linear regression operation by using weighted least square method, wherein the weight factor W is 1/X2The relative error of the measured value of each concentration point is reduced to the minimum, and the accuracy of the low-concentration sample is ensured to be the best. And (3) obtaining the back-calculated concentration and the linear back-return equation of the standard curve sample by utilizing the ratio of the peak area of the poloxamer 188 to be detected to the peak area of the internal standard through the calculation of quantitative analysis software Analyst 1.7.1. The calculated concentration from the standard curve should generally be within. + -. 15% of the indicated value, the LLOQ (lower limit of quantitation) should be within. + -. 20% and contain a minimum of 6 effective concentrations. Linear correlation coefficient r of standard curve2Should not be less than 0.98, r2The closer to 1, the better the linear relationship is illustrated. The experiment is carried out with r2The standard is not less than 0.99, so that the requirement of in vivo quantitative analysis can be met. In 3 analysis batches verified by the method, each standard curve sample consists of 7 concentration levels (0.1, 0.3, 0.5, 1.0, 3.0, 5.0 and 10.0 mug/mL), a standard curve linear regression graph is shown in FIG. 2, and a linear regression equation and related coefficients thereof are shown in Table 4 and all meet requirements.
TABLE 4 summarization of val 01-val 03 standard curve results
3. Lower limit of quantification
The Lower limit of quantitation (LLOQ) is the lowest amount of a test substance in a sample that can be quantitatively measured, and is also the lowest concentration of a standard curve, and can represent whether an analytical method has a sensitive quantitative detection capability. In chromatographic analysis, The signal-to-noise ratio (S/N) must be greater than 10 for quantification and should meet The requirements of accuracy and precision. In this test, the LLOQ was 0.1. mu.g/mL, 6 LLOQ samples were prepared, and the actual concentration of poloxamer 188 in the samples was determined, with the results shown in Table 5.
TABLE 5 LLOQ results for Poloxamer 188 in rat plasma (μ g/mL)
4. Accuracy and precision
Accuracy and precision were assessed by repeated testing of quality control samples of poloxamer 188. The accuracy is calculated by the Mean value (Mean) of the unknown sample concentration and the Deviation (DEV) of the theoretical concentration, i.e. the accuracy (%) - (Mean value of unknown sample concentration-theoretical concentration)/theoretical concentration x 100; precision was calculated from the Coefficient of Variation (CV) of unknown sample concentration, i.e., precision (%) × 100 (measured/true). The average accuracy should generally be within 15% of the indicated concentration of the quality control sample, and the variation coefficient of the precision within and between batches should generally not exceed 15%.
And (3) processing the quality control samples according to the preparation of poloxamer 188 quality control samples with labeled concentrations of 0.3, 1.5 and 8 mu g/mL, and preparing six replicates for each quality control concentration. Three analytical batches were prepared and tested in series. The results are shown in table 6, all of which meet the relevant standard specifications.
Wherein, the preparation of the quality control sample specifically comprises the following steps: the method is adopted to prepare the blank blood plasma of the rat, and the prepared poloxamer 188 quality control working solution is sequentially diluted into poloxamer 188 quality control samples (the content of the organic solvent is less than 5%) with the concentrations of 8, 1.5 and 0.3 mu g/mL.
TABLE 6 accuracy and precision of Poloxamer 188 in rat plasma
5. Matrix effect
The Matrix effect (Matrix effect) is formed by co-ionizing a non-volatile co-effluent component in a biological sample and an object to be detected at an electrospray interface, competing protons with the object to be detected, influencing the ionization efficiency of the object to be detected, causing an ion inhibition or enhancement effect, and showing an ion enhancement or inhibition effect[32,33]. The verification is carried out by analyzing and evaluating poloxamer 188 quality control samples with low, medium and high concentration levels.
The specific method comprises the following steps: randomly selecting 6 rat blank plasmas from different sources, adding poloxamer 188 and internal standard working solution into blank plasma samples to prepare quality control samples (0.3, 1.5 and 8 mu g/mL) corresponding to concentration levels, and carrying out LC-MS/MS quantitative analysis on each concentration level in a parallel mode 6 to obtain a chromatographic peak area containing a biological matrix, wherein the chromatographic peak area is named as spikematrix. Pure solution quality control samples (0.3, 1.5 and 8 mu g/mL) with corresponding concentration levels without the biological matrix are prepared, each concentration level is 6 in parallel, LC-MS/MS quantitative analysis is carried out, and the chromatographic peak area without the biological matrix is obtained and named as neratsolution. The variation coefficient of the matrix factor after internal standard normalization is not more than 15%.
Matrix Effect (spiked matrix sample peak area/fresh solution sample peak area) x 100%
The matrix effect test results are shown in table 7, and the matrix effect of the quality control sample of poloxamer 188 at three concentration levels is 82.64% -91.93%, and the variation coefficient is 5.32% -6.71%. Meets the investigation standard of matrix effect.
Table 7 matrix effect of poloxamer 188 in rat plasma (n ═ 6)
6. Extraction recovery rate
The Extraction recovery rate (Extraction recovery) is the ratio of the measured amount of the biological sample after certain pretreatment to the theoretical addition amount, and the high or low of the Extraction recovery rate can indicate whether the pretreatment method is suitable for the biological sample. The verification is also carried out by analyzing and evaluating poloxamer 188 quality control samples with low, medium and high concentration levels.
Preparing quality control samples (0.3, 1.5 and 8 mu g/mL) with three concentration levels, wherein each concentration level is 6 in parallel, processing the quality control samples, and performing LC-MS/MS quantitative analysis to obtain the chromatographic peak area of the quality control samples. The chromatographic peak area neat solution was prepared without biomatrix. The variation coefficient of the matrix factor after internal standard normalization is not more than 15%.
The extraction recovery rate (peak area of quality control sample/peak area of neat solution sample) is 100%
The result of the extraction recovery rate test is shown in table 8, the extraction recovery rate of the poloxamer 188 in the quality control samples with three concentration levels is 72.88-90.57%, and the variation coefficient is 5.52-9.11%. Meets the investigation standard of extraction recovery rate.
Table 8 recovery of poloxamer 188 from rat plasma (n ═ 6)
7. Residual investigation
After a high concentration sample is detected, a certain amount of sample is easily left in a chromatographic column or a system, and therefore, whether the sample remains in the detection process or not should be examined. According to the regulations, the residue in the blank sample should not exceed 20% of LLOQ and not exceed 5% of the internal standard after the detection of the high concentration sample. This validation required the preparation of a blank sample (carry over) and the highest concentration sample of the standard curve, i.e., the ULOQ sample, each in six replicates, and the ULOQ sample was prepared, both samples processed according to the rat plasma sample processing method. In an analysis batch, a carry over sample is detected immediately after the detection of the ULOQ sample, and whether a residual effect exists is judged according to the response values of the poloxamer 188 to be detected and the internal standard in the carry over sample. The results are shown in Table 9.
TABLE 9 residual investigation of Poloxamer 188 samples
8. Stability survey
In the actual detection and analysis process, in order to ensure that the object to be detected does not change due to changes of temperature, processing time and storage materials under the detection condition, a certain stability investigation test needs to be performed on the object to be detected so as to ensure that each link in the analysis process has no influence on the object to be detected.
(1) Stability of plasma samples after standing for 4h at room temperature before treatment
LQC (0.3 μ g/mL) and HQC (8 μ g/mL) samples were prepared as poloxamer 188 quality control samples and named LRT and HRT in the assay, each at concentration level in triplicate, and left at room temperature for 4 h. Sample treatment was performed according to the rat plasma sample treatment method. According to relevant regulations, the average value of the actual concentration of the LRT and HRT samples is within +/-15% of that of LQC and HQC, and the coefficient of variation is required to be less than 15%. As shown in table 10, the results indicate that the plasma samples were stable for 4h at room temperature prior to treatment.
TABLE 10 stability of Poloxamer 188 plasma samples prior to treatment by standing at room temperature for 4h
(2) Stability of plasma sample after being placed for 4h at room temperature
LQC (0.3 μ g/mL) and HQC (8 μ g/mL) samples were prepared as poloxamer 188 quality control samples and designated LST and HST in the assay, with triplicate samples for each concentration level. The sample treatment was performed according to the rat plasma sample treatment method, and the treated sample was left at room temperature for 4 hours. According to relevant regulations, the average value of the actual concentration of the LST and HST samples is within +/-15% of that of the LQC and HQC, and the coefficient of variation is required to be less than 15%. As shown in table 11, the results show that the plasma samples were stable after treatment for 4h at room temperature.
TABLE 11 stability of Poloxamer 188 plasma samples after treatment at room temperature for 4h
(3) Stability of plasma samples after treatment at 15 ℃ autosampler temperature for 8h
The temperature of the autosampler chamber for this test was set at 15 ℃. LQC (0.3 μ g/mL) and HQC (8 μ g/mL) samples were prepared according to poloxamer 188 quality control samples and designated LASST and HASST in the assay, with triplicate samples for each concentration level. The sample treatment was carried out according to the rat plasma sample treatment method, and immediately after the treatment, the sample was placed in an automatic sample chamber at 15 ℃ and left for 8 hours. According to relevant regulations, the actual concentration of LASST and HAST samples should be within + -15% of the deviation of LQC and HQC, and the coefficient of variation should be less than 15%. As shown in table 12, the results show that the plasma samples were stable after processing at 15 ℃ autosampler temperature for 8 h.
TABLE 12 Poloxamer 188 plasma samples post-treatment
Stability of standing at 15 ℃ autosampler temperature for 8h
(4) Repeated freeze-thaw stability of plasma sample at-20 DEG C
The test samples were stored in a-20 ℃ freezer and some samples may be subjected to multiple analyses during the assay. LQC (0.3 μ g/mL) and HQC (8 μ g/mL) samples were prepared according to the table poloxamer 188 quality control samples and designated LFT and HFT in the assay, with triplicate samples for each concentration level. The sample was processed according to the rat plasma sample processing method, and then stored in a refrigerator at-20 ℃ for at least 24 hours and thawed at room temperature. After the sample is completely thawed, the thawed plasma sample is placed into a refrigerator at-20 ℃ again for at least 12 hours each time for three freezing and thawing processes. According to relevant regulations, the mean value of the actual concentrations of LFT and HFT samples should be within + -15% of that of LQC and HQC, and the coefficient of variation should be less than 15%. As shown in Table 13, the results indicate that the plasma samples were stable by repeated freeze-thawing three times at-20 ℃.
TABLE 13 Poloxamer 188 plasma samples repeated freeze thaw stability at-20 deg.C
Comparative experiment
1. The mobile phase A ' is mixed with isopropanol in proportion, and a small amount of formic acid (acetonitrile and isopropanol of 0.1 percent formic acid are used as the mobile phase B, the volume ratio of the acetonitrile to the isopropanol is 2:3) ' is added into the mobile phase A ' instead of ' an organic phase of 0.1 percent formic acid is added into pure acetonitrile ';
experiments show that the elution strength of acetonitrile can not completely elute poloxamer 188, the elution effect is strong, and the peak pattern is poor.
2. The Agilent PLRP-S Reversed-Phase chromatographic column is replaced by a chromatographic column with models of Venusil ASB C8, SB-C18 and Extend-C18;
experiments show that the reversed phase chromatographic columns can not well separate analytes from other interference components and have the problems of serious residue, overhigh baseline, poor peak shape and the like.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein without departing from the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for LC-HR-MS/MS quantitative analysis of poloxamer, which is characterized by comprising the following steps:
1) respectively preparing a sample solution to be detected and a standard sample solution; adding an internal standard working solution into the sample solution to be detected and the standard sample solution;
2) carrying out liquid chromatography-mass spectrometry detection analysis on the standard sample solution and the sample solution to be detected;
the high performance liquid chromatography adopts a PLRP-S Reversed-Phase Column chromatography, takes 0.1-0.3% formic acid water solution as a mobile Phase A Phase, takes 0.1-0.3% formic acid acetonitrile and isopropanol as a mobile Phase B Phase, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; gradient elution was performed as follows:
0-1.4 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
1.4-1.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 40% to 65%;
1.5-3.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 65%;
3.0-3.1 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 65% to 95%;
3.1-4.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is 95%;
4.5-4.6 min: the percentage of the mobile phase B in the total volume of the mobile phase is reduced to 40 percent from 90 percent;
4.6-6.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
wherein, the condition parameters of the mass spectrometry are as follows: ion spray voltage: 5000-6000V, ion source temperature: 450-550 ℃, collision energy: 40 to 50 eV;
3) and calculating the content of the poloxamer in the sample solution to be detected.
2. The LC-HR-MS/MS quantitative analysis method according to claim 1, wherein said poloxamer is poloxamer 188.
3. The LC-HR-MS/MS quantitative analysis method according to claim 1 or 2, wherein a sample in said sample solution to be detected is a solution to be detected of plasma or an organ; and treating the solution to be detected by adopting a protein precipitation method to obtain the sample solution to be detected.
4. The LC-HR-MS/MS quantitative analysis method according to claim 3, wherein said precipitated protein method treatment is specifically: acetonitrile and isopropanol of 0.1-0.3% formic acid are used as precipitation reagents, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; adding a precipitation reagent into the liquid to be detected according to the volume ratio of 3-5: 1 to precipitate protein; centrifuging at high speed after vortexing, and taking supernate to obtain the sample solution to be detected;
preferably, the organ further comprises a pretreatment, specifically: and (4) stirring the in vitro viscera to homogenate, and centrifuging at a high speed to obtain a supernatant fluid to be detected.
5. The LC-HR-MS/MS quantitative analysis method according to any one of claims 1 to 4, wherein the internal standard working solution is a simvastatin solution with the volume ratio of 0.8 to 1.2 μ g/mL, and the solvent of the simvastatin solution is acetonitrile and water with the volume ratio of 2:2.5 to 3.5;
preferably, the volume ratio of the sample solution to be detected to the internal standard working solution is 1: 0.8-1.2; the volume ratio of the standard sample solution to the internal standard working solution is 1: 0.8-1.2.
6. The LC-HR-MS/MS quantitative analysis method according to any one of claims 1 to 5, wherein said standard solution is prepared by the following method:
preparing at least 6 gradient poloxamer standard solutions of 2.5-250 mug/mL; the solvent of the poloxamer standard solution is acetonitrile and water in a volume ratio of 2: 2.5-3.5.
7. The LC-HR-MS/MS quantitative analysis method according to any one of claims 1 to 6, wherein said conditions of high performance liquid chromatography further comprise at least one of:
the column temperature is 35-45 ℃,
the flow rate is 0.7-0.9 mL/min;
the temperature of the automatic sample injector is 12-18 ℃;
preferably, the conditions of the high performance liquid chromatography further include at least one of:
taking a 0.1% formic acid aqueous solution as a mobile phase A phase, taking 0.1% formic acid acetonitrile and isopropanol as a mobile phase B phase, wherein the volume ratio of the acetonitrile to the isopropanol is 2: 3;
the column temperature was 40 c,
the flow rate is 0.8 mL/min;
autosampler temperature 15 ℃.
8. The LC-HR-MS/MS quantitative analysis method according to any one of claims 1 to 7, wherein said mass spectrometry conditions further comprise at least one of:
air curtain air: 30-40 psi;
GS1:35～55psi；
GS2:35～55psi；
cluster-splitting voltage: 90-110V;
collision energy: 40 to 50 eV;
MSALLscanning range: 50-1250 Da;
ion extraction window width for extracting poloxamer 188 characteristic fragment ions: 0.01 Da;
preferably, the conditions of mass spectrometry are: ion spray voltage: 5500V, ion source temperature: 500 ℃, gas curtain gas: 35psi, GS1:40psi, GS2:40psi, declustering voltage: 100V, collision energy: 45eV, MSALL scan range: 50-1250Da, and the width of an ion extraction window for extracting poloxamer 188 characteristic fragment ions: 0.01 Da.
9. The LC-HR-MS/MS quantitative analysis method according to any one of claims 1 to 8, wherein the step 3) is specifically:
performing LC-MS/MS quantitative analysis on a standard sample solution, performing linear regression operation by using a weighted least square method according to the peak area ratio of the standard sample solution to an internal standard substance, wherein a weight factor W is 1/X2(ii) a Calculating by quantitative analysis software Analyst 1.7.1 to obtain the back-calculated concentration and linear regression equation of the standard curve sample; and comparing the peak area of the solution with the peak area of the sample to be detected, and calculating the content of poloxamer in the solution of the sample to be detected based on the linear regression equation.
10. The LC-MS/MS quantitative analysis of any one of claims 1 to 9, comprising the steps of:
1) preparing a sample solution to be tested: treating the solution to be detected by adopting a protein precipitation method; specifically, the method comprises the following steps: acetonitrile and isopropanol of 0.1-0.3% formic acid are used as precipitation reagents, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; adding a precipitation reagent into the liquid to be detected according to the volume ratio of 3-5: 1, and precipitating protein; centrifuging at high speed after vortexing, and taking supernate to obtain the sample solution to be detected;
wherein the liquid to be detected is blood plasma to be detected or organ to be detected after pretreatment; the pretreatment specifically comprises the following steps: crushing the in vitro viscera into homogenate, and centrifuging at high speed to obtain supernatant fluid to obtain fluid to be detected;
2) preparation of standard sample solutions: preparing at least 6 gradient poloxamer 188 standard solutions of 2.5-250 mug/mL; the solvent of the poloxamer standard solution is acetonitrile and water in a volume ratio of 2: 2.5-3.5;
adding an internal standard working solution into the to-be-detected sample solution and the standard sample solution according to the volume ratio of 1: 0.8-1.2 respectively; the internal standard working solution is a simvastatin solution with the volume ratio of 0.8-1.2 mu g/mL, and the solvent of the simvastatin solution is acetonitrile and water with the volume ratio of 2: 2.5-3.5;
3) carrying out liquid chromatography-mass spectrometry detection analysis on the standard sample solution and the sample solution to be detected;
the high performance liquid chromatography adopts a PLRP-S Reversed-Phase Column chromatography, takes 0.1-0.3% formic acid water solution as a mobile Phase A Phase, takes 0.1-0.3% formic acid acetonitrile and isopropanol as a mobile Phase B Phase, and the volume ratio of the acetonitrile to the isopropanol is 2: 3-4; gradient elution was performed as follows:
0-1.4 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
1.4-1.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 40% to 65%;
1.5-3.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 65%;
3.0-3.1 min: the percentage of the mobile phase B in the total volume of the mobile phase is increased from 65% to 95%;
3.1-4.5 min: the percentage of the mobile phase B in the total volume of the mobile phase is 95%;
4.5-4.6 min: the percentage of the mobile phase B in the total volume of the mobile phase is reduced to 40 percent from 90 percent;
4.6-6.0 min: the percentage of the mobile phase B in the total volume of the mobile phase is 40%;
the column temperature is 35-45 ℃, and the flow rate is 0.7-0.9 mL/min; the temperature of the automatic sample injector is 12-18 ℃;
wherein, the condition parameters of the mass spectrometry are as follows: ion spray voltage: 5000-6000V, ion source temperature: 450-550 ℃, collision energy: 40 to 50 eV; air curtain air: 30-40 psi; GS1, 35-45 psi; GS2, 35-45 psi; cluster-splitting voltage: 90-110V; collision energy: 40 to 50 eV; MS (Mass Spectrometry)ALLScanning range: 50-1250 Da; ion extraction window width for extracting poloxamer 188 characteristic fragment ions: 0.01 Da;
4) performing LC-MS/MS quantitative analysis, performing linear regression by using the peak area ratio of the standard sample solution to the internal standard substance and adopting a weighted least square method, wherein the weight factor W is 1/X2(ii) a Calculating by quantitative analysis software Analyst 1.7.1 to obtain the back-calculated concentration and linear regression equation of the standard curve sample; then comparing with the peak area of the sample solution to be measured based onAnd (5) calculating the content of poloxamer in the sample solution to be detected by using a linear regression equation.
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