CN107367562B - Analysis and detection method and application of polymyxin B sulfate - Google Patents

Analysis and detection method and application of polymyxin B sulfate Download PDF

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CN107367562B
CN107367562B CN201710656465.6A CN201710656465A CN107367562B CN 107367562 B CN107367562 B CN 107367562B CN 201710656465 A CN201710656465 A CN 201710656465A CN 107367562 B CN107367562 B CN 107367562B
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CN107367562A (en
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贾存宇
李晓倩
陆倩
黄臻辉
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Add Medicine To First Biochemical Pharmaceutcal Corp Ltd In Shanghai
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Abstract

The invention discloses an analysis and detection method of polymyxin B sulfate. The detection method comprises the following steps: and (3) analyzing and detecting polymyxin B sulfate by adopting high performance liquid chromatography combined with an evaporation light diffuser. The method realizes complete separation of four components in the polymyxin B sulfate, has strong specificity and simple operation, is not interfered by other organic impurities, has the effect similar to that of an HPLC method of EP 9.0 bulk drug, has high sensitivity, is simple and quick, and can be more accurately used for determining the content of each component in the polymyxin B sulfate preparation.

Description

Analysis and detection method and application of polymyxin B sulfate
Technical Field
The invention relates to a polymyxin B sulfate analysis and detection method and application.
Background
Polymyxin b (polymyxin b) is a basic polypeptide antibiotic produced by bacillus polymyxa and composed of various amino acids and fatty acids. The current production is mainly obtained by biosynthesis by adopting a microbial fermentation method and then by extraction and refining. The sulfate is used clinically as anti-infective medicine for treating skin wound, urinary system infection, eye infection, ear infection, trachea infection, septicemia, peritonitis, etc. caused by gram-negative bacteria such as Pseudomonas aeruginosa. Polymyxin B sulfate is a multi-component mixture with similar structure, and comprises polymyxin B1、B2、B3、B1-I. Wherein, B1And B2Is an active ingredient, B3And B1-I is the main impurity. The chemical structural formula is as follows:
Figure BDA0001369408740000011
specifically, polymyxin B1、B2、B3And B1The structural differences of-I are as follows:
Figure BDA0001369408740000012
Figure BDA0001369408740000021
at present, the related analysis and detection methods of polymyxin B sulfate or preparations thereof are recorded in european pharmacopoeia EP 9.0, united states pharmacopoeia USP40 and chinese pharmacopoeia ChP 2015.
Wherein, in EP 9.0, the content of polymyxin B sulfate is determined by isocratic elution by reversed phase high performance liquid chromatography of a sodium sulfate-phosphoric acid-water-acetonitrile system, ultraviolet wavelength is detected at 215nm, the content of each component is calculated by an external standard method, and the single-needle analysis time is about 50 min. A method of detection without a relevant agent;
although USP40 and ChP 2015 adopt a microbial antibiotic titer method to calculate the content of polymyxin B sulfate and preparations thereof, high performance liquid chromatography which is consistent with EP 9.0 is also adopted under a polymyxin component inspection item, and the total content of B1, B2, B3 and B1-I is more than or equal to 80.0%, B3 is less than or equal to 6.0% and B1-I is less than or equal to 15.0% in terms of dry products;
orwa et al examined polymyxin B sulfate at various pH's at 215nm using acetonitrile-sodium sulfate aqueous solution (0.7%, m/v) -phosphoric acid aqueous solution (5.8%, v/v) -water (22.25: 50: 5: 22.75) as the base mobile phase1、B2、B3The isolation of (Journal of Chromatography A,870(2000) 237-.
Due to the similarity of the structures of the components in polymyxin B sulfate, the components only differ in the fatty acid chain and the amino acid residue at the Z position, and the content difference of each single component is large. In addition, polymyxin B sulfate absorbs weakly at 215nm, and the signal intensity of each component is low when the method is adopted, so that the accuracy of chromatographic integration in quantitative analysis is influenced to a certain extent; in addition, the isocratic elution method also has the disadvantages of low column efficiency, long analysis time and the like for separating each component. Therefore, the development of a method which is rapid, sensitive and accurate and can simultaneously carry out quantitative analysis on each component has great significance on the quality control of the medicine.
Disclosure of Invention
The invention solves the technical problems that the specificity is poor and the precision is low when the polymyxin B sulfate is measured by adopting an antibiotic microbial titer method or the problems of complex flow phase preparation, low sensitivity, poor repeatability, long analysis time and the like exist when the polymyxin B sulfate is analyzed and detected by adopting a high performance liquid chromatography-ultraviolet detector in the prior art, so that the invention provides the method for analyzing and detecting the polymyxin B sulfate and the application thereof.
The invention solves the technical problems through the following technical scheme.
The invention provides an analysis and detection method of polymyxin B sulfate, which comprises the following steps: analyzing and detecting polymyxin B sulfate by adopting a high performance liquid chromatography combined evaporation light diffuser;
the chromatographic column in the high performance liquid chromatography is a reversed phase analytical column, and the stationary phase of the chromatographic column is octadecyl bonded silica gel; such as a Kromasil 100-5C18 column;
the mobile phase in the high performance liquid chromatography is a polar organic solvent and a trifluoroacetic acid aqueous solution, and the volume percentage content of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01-0.3%; the volume percentage of the trifluoroacetic acid in the aqueous trifluoroacetic acid solution is calculated by the volume of the trifluoroacetic acid in the total volume of the aqueous trifluoroacetic acid solution.
In the invention, the polymyxin B sulfate is polymyxin B1、B2、B3And B1-I mixture.
In the invention, the high performance liquid chromatography can adopt gradient elution, and the volume ratio of the trifluoroacetic acid aqueous solution to the polar organic solvent during the gradient elution can be 85: 15-60: 40.
In the present invention, the range of the flow phase ratio over time in the gradient elution can be shown in table 1:
TABLE 1
Figure BDA0001369408740000031
Figure BDA0001369408740000041
Further, it can be shown in table 2 or table 3:
TABLE 2
Time (min) Polar organic solvent (%, v/v)
0 24
5.00 24
5.01～8.00 24～29
8.01～15.00 29～32
15.01～20.00 32～40
TABLE 3
Time (min) Polar organic solvent (%, v/v)
0 24
5.00 24
5.01～8.01 24～27
8.01～15.00 27～30
15.01～20.00 30～40
In the invention, the trifluoroacetic acid in the trifluoroacetic acid aqueous solution can be 0.05-0.1% by volume.
In the present invention, the polar organic solvent may employ one or more of polar organic solvents commonly used in high performance liquid chromatography in the art, such as methanol, ethanol, and acetonitrile.
In the present invention, the hplc in the hplc can adopt a conventional hplc in the art, for example, a model of Alliance e2695 hplc from Waters.
In the present invention, the specification of the chromatographic column may be the specification of analytical columns conventional in the art, and the following specifications of chromatographic columns may be selected: the filler particle size of the chromatographic column can be 2.7-5 μm; for example, 3 μm to 5 μm; the length of the chromatographic column may be from 100mm to 250mm, for example 150 mm; the sample injection volume of the chromatographic column can be 1-50 mu L, such as 20 mu L.
In the invention, the column temperature in the high performance liquid chromatography can be the conventional temperature of the operation in the field, such as 20-40 ℃, and further such as 35 ℃.
In the present invention, the flow rate of the mobile phase can be determined by referring to the conventional operation in the field, such as 0.5-1.5 mL/min, and further such as 0.8-1.0 mL/min.
In the present invention, the evaporation light diffuser may be an evaporation light diffuser used with a hplc, such as an evaporation light diffuser manufactured by Waters model 2424, and the parameters of the evaporation light diffuser may be set according to conventional settings, such as:
the drift tube temperature of the evaporation light diffuser can be 40-80 ℃; for example, 60 to 70 ℃;
the carrier gas pressure of the evaporative light diffuser may be 20 to 45 psi; such as 25 to 35 psi;
the temperature of the atomizing opening of the evaporation light diffuser can be 12-35 ℃; for example 12 to 30 ℃.
In the invention, the polymyxin B sulfate can be replaced by a raw material medicine of the polymyxin B sulfate, or a preparation of the polymyxin B sulfate, or a mixture of two or more than two components B1, B2, B3 and B1-I in the polymyxin B sulfate.
The invention also provides application of the analysis and detection method in content detection of polymyxin B sulfate bulk drug or preparation, which comprises the following steps:
step (1), respectively preparing a reference substance solution and a polymyxin B sulfate bulk drug or preparation to-be-detected substance solution with certain concentrations, and analyzing and detecting the reference substance solution and the polymyxin B sulfate preparation to-be-detected substance solution by adopting the analysis and detection method; measuring peak areas of each component in the reference substance solution and the polymyxin B sulfate preparation;
step (2), obtaining a regression equation according to the peak area and the corresponding component concentration of each component in the polymyxin B sulfate in the reference solution;
and (3) substituting the peak areas of the components measured in the step (1) into the regression equation obtained in the step (2) to obtain the content of each component of the polymyxin B sulfate bulk drug or preparation.
The formulation may be a sterile powder for injection.
In the step (1), the reference substance may be polymyxin B sulfate USP reference substance, for example, having a lot number of N1M425 and containing B per 100mg of the reference substance169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg；
In the step (1), the reference solution and the polymyxin B sulfate preparation solution to be tested can be prepared by using a conventional solvent capable of dissolving the reference solution and the polymyxin B sulfate solution, for example, a mixed solvent of acetonitrile and water in a volume ratio of 8: 2;
the concentration of the reference solution can refer to the concentration of the field when a double logarithmic regression equation of the concentration and the peak area is made, such as 0.3 mg/mL-1.5 mg/mL; the concentration of the polymyxin B sulfate preparation test solution can be selected according to the concentration range of the reference substance, such as 0.5 mg/mL.
In the step (2), the regression equation may be obtained by referring to a conventional calculation method in the art, for example, by using a logarithm of the peak area of each component in the polymyxin B sulfate in the control solution with 10 as a base as an abscissa and a logarithm of the mass concentration of the corresponding component with 10 as a base as an ordinate, fitting a quadratic curve, and calculating the regression equation.
In the step (3), the contents of the components of the polymyxin B sulfate preparation can be substituted into the regression equation obtained in the step (2) by taking the logarithm with the base of 10 of the peak area of each component measured in the step (1).
The application of the analysis and detection method in the content detection of the polymyxin B sulfate preparation comprises the following steps:
(1) weighing a proper amount of polymyxin B sulfate reference substance (which is a mixture with known contents of all components, taking USP reference substance as an example, every 100mg contains B169.7mg, B215.9mg, B33.2mg and B1-I6.1 mg), placing the polymyxin B sulfate reference substance into a volumetric flask, dissolving the polymyxin B sulfate reference substance with water-acetonitrile (80:20) as a solvent, and diluting the solution to a scale to obtain a solution with the total concentration of 5.0mg/mL of polymyxin B sulfate, which is used as a reference substance solution A; taking a proper amount from the solution A, respectively placing the solution A into volumetric flasks, and preparing reference substance solutions with the total concentrations of 0.3 mg/mL-1.5 mg/mL respectively;
taking a proper amount of polymyxin B sulfate bulk drug or preparation sample, and preparing a sample solution with the concentration of 0.5mg/mL by taking water-acetonitrile (80:20, volume ratio) as a solvent;
(2) sampling 20 mu L of each of the reference solution and the test solution, and fitting a quadratic curve after respectively taking logarithms according to the peak area of each component of polymyxin B sulfate in the reference solution and the mass concentration of the corresponding solution component by adopting the analysis and detection method;
(3) the content of each component in the test solution is calculated by substituting the logarithm of the measured peak area into a standard curve; the sum of the contents of the components of polymyxin B sulfate is the total content.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and apparatus used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the analysis and detection method has the characteristics of simple operation, strong specificity, high sensitivity, simplicity, rapidness and the like.
(2) The analysis and detection method has good linearity of the logarithm of the concentration-the logarithm of the peak area of each component of the polymyxin B in the quality control range specified by pharmacopoeia of various countries;
(3) the analysis and detection method is suitable for identification or quantitative detection of each component in the polymyxin B sulfate, can also be used for content detection of the polymyxin B sulfate in a polymyxin B sulfate raw material medicine or a preparation, or is further used for monitoring the preparation process of the polymyxin B sulfate, the raw material medicine or the preparation, and is high in practicability.
Drawings
FIG. 1 is a HPLC chart of the analytical detection method of polymyxin B sulfate in example 1;
FIGS. 2 to 5 are graphs of concentration-peak area log fitting curves of components of polymyxin B sulfate in example 2;
FIG. 6 is an HPLC chart of content detection of each component in the polymyxin B sulfate bulk drug in example 3;
FIG. 7 is an HPLC chart showing content detection of each component in polymyxin B sulfate for injection in example 4;
FIG. 8 is an HPLC chart showing content measurement of each component in polymyxin B sulfate for injection in example 5;
FIG. 9 is an HPLC chart showing content measurement of each component in polymyxin B sulfate for injection in example 6;
FIG. 10 is a HPLC chart of the analytical detection method of polymyxin B sulfate in comparative example 1;
FIG. 11 is a HPLC chart of the analytical detection method of polymyxin B sulfate in comparative example 2;
FIG. 12 is a HPLC chart of the analytical detection method of polymyxin B sulfate in comparative example 3;
FIG. 13 is an HPLC chart of the analytical detection method for polymyxin B sulfate drug substance in comparative example 4;
FIG. 14 is an HPLC chart of the analytical detection method for polymyxin B sulfate drug substance in comparative example 5;
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
Specificity and sensitivity of the assay
The experimental conditions are as follows:
trifluoroacetic acid (HPLC grade, tiandi ltd, usa);
acetonitrile (HPLC grade, semer feishell science ltd);
polymyxin B sulfate USP control (batch No. N1M425 containing B per 100 mg)169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg)；
High performance liquid chromatograph: alliance e2695 high performance liquid chromatography (Waters);
evaporative light scattering detector: model 2424 evaporative light scattering detector (Waters);
an Empower 3 high performance liquid chromatograph data processing system;
a chromatographic column: kromasil 100-5C18 column (5 μm, 4.6X 250 mm);
the mobile phase A is 0.1% trifluoroacetic acid aqueous solution (pH value is 1.95), the mobile phase B is acetonitrile, the gradient change of the acetonitrile volume ratio is 0min to 5min, and the gradient change is kept at 24%; 5min to 8min, from 24% to 29%; 8min to 15min, from 29% to 32%; 15min to 20min, from 32% to 40%. The flow rate was 1.0mL/min, and the column temperature was 35 ℃.
The detector drift tube temperature was 60 ℃, the atomization orifice temperature was 12 ℃, the carrier gas pressure was 35.0psi, and the gain was 800.
Experimental procedure
50mg of a polymyxin B sulfate USP control sample is precisely weighed, placed in a 100mL volumetric flask, dissolved and diluted to the scale with water-acetonitrile (80:20) as a solvent. The sample injection is repeated 6 times with 20 μ L. And recording the chromatogram.
Calculating the logarithm of the peak area of six needles of each component to obtain RSD and polymyxin B sulfate2、B3、B1-I、B1RSD of (a) is 0.22%, 0.50%, 0.29% and 0.13% accordingly. Taking the spectrum of the first injection as an example, as shown in fig. 1, and combining table 4, it can be known that: polymyxin B sulfate2、B3、B1-I、B1The retention time of the components is 10.904min, 11.230min, 11.854min and 12.409min in sequence, the four components are completely separated, and other impurities are not interfered. The result shows that the system is precise and good, and the method has good specificity on each component of polymyxin B sulfate.
TABLE 4
Component name Retention time/min Peak area Degree of separation
Polymyxin B2 10.904 521591 -
Polymyxin B3 11.230 38276 1.9
Polymyxin B1-I 11.854 101355 3.8
Polymyxin B1 12.409 5242581 3.1
In addition, the same reference substance solution is taken, and the injection detection is carried out by adopting the method under the inspection item of the polymyxin B component in USP40, and the result shows that polymyxin B is removed1In addition, the response value of each component is less than 50 mAU. The signal to noise ratio and theoretical plate number of the analytical detection method of this method and USP40 were compared, and the results are shown in table 5:
TABLE 5
Figure BDA0001369408740000091
The result shows that the detection sensitivity and the column efficiency of each component of the polymyxin B sulfate with larger relative content difference in the method are obviously improved, and the method is favorable for improving the integration accuracy of the chromatographic peak area.
Example 2
Linearity and range of analytical methods
Conditions of the experiment
Experiments were performed using similar experimental conditions as in example 1. Polymyxin B sulfate USP control (lot)Number N1M425, each 100mg containing B169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg)。
Experimental procedure
100mg of polymyxin B sulfate USP reference substance is precisely weighed, placed in a 20mL volumetric flask, dissolved and diluted to a scale by using water-acetonitrile (80:20) as a solvent, 600 muL, 800 muL, 900 muL, 1000 muL, 1100 muL, 1200 muL, 1400 muL, 2000 muL and 3000 muL of the mother solution are sucked and respectively placed in a 10mL volumetric flask, and the reference solution is used after dilution and volume fixing. And (4) sampling 20 mu L of the solution, and recording a chromatogram. And fitting a quadratic curve by taking the logarithm of the peak area of each component with the base 10 as the abscissa and the logarithm of the mass concentration of the corresponding component with the base 10 as the ordinate.
Calculated, the polymyxin B is in the range of 0.0457 mg/mL-0.165 mg/mL2Is given as y-3.4464 x2–4.7041x+4.7527，R20.9978; within the range of 0.0092 mg/mL-0.0499 mg/mL of concentration, polymyxin B3Is given as y-1.8517 x2–3.5x+4.3049，R20.9959; in the concentration range of 0.0175 mg/mL-0.0951 mg/mL, polymyxin B1-The regression equation for I is y-2.2471 x2–3.5297x+4.8556，R20.9964; in the concentration range of 0.2003 mg/mL-0.4673 mg/mL, polymyxin B1Has a regression equation of-2.773 x2–0.0979x+7.3827，R20.9980. As shown in fig. 2 to 5.
The result shows that the method has good linearity in the quality control range specified by pharmacopoeia of each country for the concentration logarithm-peak area logarithm of each component of polymyxin B.
Example 3
Content detection of each component in polymyxin sulfate B bulk drug
Conditions of the experiment
Experiments were performed using similar experimental conditions as in example 1. The difference is only that: the gradient change of the acetonitrile volume ratio is 0min to 5min, and is kept at 24 percent; 5min to 8min, from 24% to 27%; 8min to 15min, from 27% to 30%; 15min to 20min, from 30% up to40 percent. The carrier gas pressure was 30 psi. The reference polymyxin B sulfate USP has a lot number of N1M425, and each 100mg contains B169.7mg、B215.9mg、B33.2mg、B1-I6.1 mg. Polymyxin B sulfate drug substance lot a1411105 was purchased from xella pharmaceutical, denmark.
Experimental procedure
50mg of a polymyxin B sulfate USP reference substance is precisely weighed, placed in a 10mL volumetric flask, dissolved and diluted to a scale by using water-acetonitrile (80:20) as a solvent, 600 muL, 900 muL, 1000 muL, 1100 muL, 1400 muL, 2000 muL and 3000 muL of the mother solution are sucked and respectively placed in the 10mL volumetric flask, and the reference solution is obtained after dilution and volume fixing. 50mg of polymyxin B sulfate is precisely weighed, placed in a 100Ml volumetric flask, dissolved and diluted to a scale with water-acetonitrile (80:20) as a solvent to be used as a test solution. And (4) sampling 20 mu L of the solution, and recording a chromatogram. And fitting a quadratic curve by taking the logarithm of the peak area of each component of the control solution with the base 10 as the abscissa and the logarithm of the mass concentration of the corresponding component with the base 10 as the ordinate. The content of each component in the test sample is calculated by substituting the logarithm of the measured peak area into the standard curve. The sum of the contents of the components of polymyxin B sulfate is the total content.
As shown in FIG. 6, the separation degree of each component of polymyxin B was good, and polymyxin B was2Peak and B3Peak separation degree of 2.1, polymyxin B3Peak and B1Resolution of the I peak 3.9, polymyxin B1-peak I and B1The peak separation was 2.8. Calculated, polymyxin B2、B3、B1-I、B1The content of (A) was 64.4%, 11.2%, 3.7% and 9.2%, respectively, and the content of polymyxin B was 88.5%.
Example 4
Content detection of polymyxin B sulfate for injection
Conditions of the experiment
An experiment was conducted using similar experimental conditions and procedures as in example 3, wherein the aqueous phase in the mobile phase was replaced with 0.1% by volume aqueous trifluoroacetic acid. The temperature of the atomizing opening is 30 ℃, and the temperature of the drift tube is 70 ℃. Polymyxin B sulfate for injection was made in house, lot number 1507801.
As shown in FIG. 7, the separation degree of each component of polymyxin B was good, and polymyxin B was2Peak and B3Peak separation degree of 1.8, polymyxin B3Peak and B1Resolution of the I peak 3.8, polymyxin B1-peak I and B1The peak separation was 3.2. Calculated, polymyxin B2、B3、B1-I、B1The content of (A) is 64.5%, 11.0%, 3.4% and 7.3%, respectively, and the content of polymyxin B is 86.2%.
Example 5
An experiment was conducted using similar experimental conditions and procedures as in example 1, wherein the aqueous phase in the mobile phase was replaced with 0.01% by volume aqueous trifluoroacetic acid. The reference polymyxin B sulfate USP has a lot number of N1M425, and each 100mg contains B169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg。
As shown in FIG. 8, the separation degree of each component of polymyxin B was good, and polymyxin B was2Peak and B3Peak separation degree of 2.1, polymyxin B3Peak and B1Resolution of the I peak 3.1, polymyxin B1-peak I and B1The peak separation was 2.0. With polymyxin B1Peak meter, theoretical plate number 18243, signal to noise ratio S/N1149.
Example 6
An experiment was conducted using similar experimental conditions and procedures as in example 1, wherein the aqueous phase in the mobile phase was replaced with 0.01% by volume aqueous trifluoroacetic acid. The reference polymyxin B sulfate USP has a lot number of N1M425, and each 100mg contains B169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg。
As shown in FIG. 9, the separation degree of each component of polymyxin B was good, and polymyxin B was2Peak and B3Peak separation degree of 2.1, polymyxin B3Peak and B1Resolution of the I peak 3.1, polymyxin B1-peak I and B1The peak separation was 2.0. With polymyxin B1Peak meter, theoretical plate number 18243, Signal-to-noise ratioThe S/N is 1149.
Comparative example 1
An experiment was conducted using similar experimental conditions and procedures as in example 1, wherein the aqueous phase in the mobile phase was replaced with 0.01% by volume aqueous trifluoroacetic acid. The reference polymyxin B sulfate USP has a lot number of N1M425, and each 100mg contains B169.7mg、B215.9mg、B33.2mg、B1-I 6.1mg。
As shown in FIG. 10, the separation degree of each component of polymyxin B was good, and polymyxin B was2Peak and B3Peak separation degree of 2.1, polymyxin B3Peak and B1Resolution of the I peak 3.1, polymyxin B1-peak I and B1The peak separation was 2.0. With polymyxin B1Peak meter, theoretical plate number 18243, signal to noise ratio S/N1149.
Comparative example 2
Reference example 1, except that the aqueous phase was 20mmol/L ammonium formate (pH 2.1) in water, polymyxin B could not be added1-I and B1And (4) separating. As shown in fig. 11, the components are poorly retained and cannot be separated.
Comparative example 3
Reference example 1, except that the aqueous phase was 0.1% formic acid, the components of polymyxin B could not be separated. As shown in fig. 12, the components are poorly retained and cannot be separated.
Comparative example 4
In this comparative example, the content of polymyxin B sulfate was determined using similar experimental conditions and procedures as in example 3, except that the aqueous phase was 0.1% formic acid; kromasil C18 (4.6X 250mm,5 μm) was used; the gradient change of the acetonitrile volume ratio is 0min to 5min, and is kept at 12 percent; 5min to 8min, from 12% to 20%; 8min to 15min, from 20% to 30%; 15min to 20min, from 30% to 40%. (ii) a The drift tube temperature of the detector used for evaporative light scattering was 70 ℃; the flow rate of the mobile phase is 0.8 ml/min; the drift tube temperature of the detector used for evaporative light scattering was 40 ℃.
As shown in FIG. 13, the number of the parts cannot be increased in this embodimentColistin B1-I and B1And (5) separating.
Comparative example 5
In this example, the content of polymyxin B sulfate was determined under similar experimental conditions and procedures as in example 3, except that the aqueous phase was 20mM ammonium acetate aqueous solution (pH 4.6); the gradient change of the acetonitrile volume ratio is 0min to 5min, and is kept at 15 percent; 5min to 8min, from 15% to 24%; 8min to 15min, from 24% to 32%; 15min to 20min, from 32% to 40%; the drift tube temperature of the detector used for evaporative light scattering was 70 ℃; the carrier gas pressure was 35 psi.
As shown in FIG. 14, the components of polymyxin B could not be separated in this example.

Claims (11)

1. An analytical detection method of polymyxin B sulfate is characterized by comprising the following steps: analyzing and detecting polymyxin B sulfate by adopting a high performance liquid chromatography combined evaporation light diffuser;
the chromatographic column in the high performance liquid chromatography is a reversed phase analytical column, and the stationary phase of the chromatographic column is octadecyl bonded silica gel;
the mobile phase in the high performance liquid chromatography is a polar organic solvent and a trifluoroacetic acid aqueous solution, and the volume percentage content of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01-0.3%;
the high performance liquid chromatography adopts gradient elution, and the volume ratio of the trifluoroacetic acid aqueous solution to the polar organic solvent during the gradient elution is 85: 15-60: 40;
the range of the flow phase ratio over time in the gradient elution is shown in table 1:
TABLE 1
Time (min) Polar organic solvent (%, v/v) 0～5.00 15～24 5.01～8.00 24～29 8.01～15.00 27～32 15.01～20.00 30～40
。
2. The analytical method according to claim 1, wherein the range of change in the flow phase ratio with time in the gradient elution is shown in table 2 or table 3:
TABLE 2
Time (min) Polar organic solvent (%, v/v) 0 24 5.00 24 5.01～8.00 24～29 8.01～15.00 29～32 15.01～20.00 32～40
TABLE 3
Time (min) Polar organic solvent (%, v/v) 0 24 5.00 24 5.01～8.01 24～27 8.01～15.00 27～30 15.01～20.00 30～40
。
3. The analytical detection method according to claim 1, wherein the trifluoroacetic acid in the aqueous trifluoroacetic acid solution is in a volume percentage of 0.05% to 0.1%.
4. The analytical detection method according to claim 1, wherein the polar organic solvent is selected from one or more of methanol, ethanol, and acetonitrile;
and/or the high performance liquid chromatograph in the high performance liquid chromatography is a Waters high performance liquid chromatograph with the model number of Alliance e 2695;
and/or, the chromatographic column is a Kromasil 100-5C18 column;
and/or the filler particle size of the chromatographic column is 2.7-5 μm; the length of the chromatographic column is 100 mm-250 mm;
and/or the sample injection volume of the chromatographic column is 1-50 mu L;
and/or the column temperature in the high performance liquid chromatography is 20-40 ℃;
and/or the flow rate of the mobile phase is 0.5-1.5 mL/min.
5. The analytical detection method of claim 4, wherein the filler particle size of the chromatographic column is 3 μm to 5 μm; the length of the chromatographic column is 250 mm;
and/or the sample injection volume of the chromatographic column is 20 mu L;
and/or, the column temperature in the high performance liquid chromatography is 35 ℃;
and/or the flow rate of the mobile phase is 0.8-1.0 mL/min.
6. The analytical detection method of claim 1, wherein the evaporative diffuser is a Waters model 2424 evaporative diffuser;
and/or the drift tube temperature of the evaporation light diffuser is 40-80 ℃;
and/or the carrier gas pressure of the evaporation light diffuser is 20-45 psi;
and/or the temperature of the atomization opening of the evaporation light diffuser is 12-35 ℃.
7. The analytical testing method of claim 6, wherein said evaporative light diffuser has a drift tube temperature of 60 ℃ to 70 ℃;
and/or the carrier gas pressure of the evaporative light diffuser is 25-35 psi;
and/or the temperature of the atomizing opening of the evaporation light diffuser is 12-30 ℃.
8. Use of an analytical test method according to any one of claims 1 to 7 for the determination of the content of a polymyxin B sulfate drug substance or formulation.
9. The use of claim 8, wherein the formulation is a sterile powder for injection.
10. The use of claim 8, comprising the steps of: step (1), respectively preparing a reference substance solution and a polymyxin B sulfate bulk drug or preparation to-be-detected substance solution with certain concentrations, and analyzing and detecting the reference substance solution and the polymyxin B sulfate preparation to-be-detected substance solution by adopting the analysis and detection method; measuring peak areas of each component in the reference substance solution and the polymyxin B sulfate preparation;
step (2), obtaining a regression equation according to the peak area and the corresponding component concentration of each component in the polymyxin B sulfate in the reference solution;
and (3) substituting the peak areas of the components measured in the step (1) into the regression equation obtained in the step (2) to obtain the content of each component of the polymyxin B sulfate bulk drug or preparation.
11. The use of claim 10, wherein the regression equation is calculated by fitting a quadratic curve.
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