CN108152414B - Method for detecting avilamycin premix by HPLC-ELSD - Google Patents
Method for detecting avilamycin premix by HPLC-ELSD Download PDFInfo
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Abstract
The invention provides a method for detecting avilamycin premix related substances by HPLC-ELSD, which comprises the following steps: preparing an avilamycin reference substance solution and a sample solution, drawing a standard curve, detecting by adopting HPLC-ELSD, recording peak areas, and calculating the content of each component peak and impurity peak of the avilamycin in the sample by adopting a standard curve method. The method is improved on the basis of the bulletin method, and the leaching solvent is changed from acetone to methanol, so that the toxicity and the volatility are reduced; the sample processing steps are simplified, and the efficiency is higher; the separation degree between peaks is improved after the gradient elution condition is optimized, the calculation of each component is more accurate, the pH value of a mobile phase does not need to be adjusted, the time is saved, and the possibility of misoperation is reduced; the detector is changed from an ultraviolet detector to a universal evaporative light scattering detector, the detection range is wider, and the monitoring on impurities is more comprehensive and stricter.
Description
Technical Field
The invention belongs to the field of component detection, relates to a method for detecting avilamycin related substances, and particularly relates to a method for detecting avilamycin premix related substances by HPLC-ELSD.
Background
The avilamycin is produced by fermenting Streptomyces viridochromogenes and belongs to oligosaccharide antibiotics. The components of the composition comprise 14 components from A to N, wherein the avilamycin A is the main component, the physiological activity of the composition is highest, and the inhibition effect on clostridium, streptococcus and bacillus is particularly high. Followed by avilamycin B.
The requirements of the quality standard of the avilamycin premix issued by Ministry of agriculture of the people's republic of China No. 2253 on each component are as follows: the peak of the avilamycin A is not lower than 60%, the peak of the avilamycin B is not higher than 18%, the peak of the avilamycin A and the peak of the avilamycin B are not lower than 70%, and other single components are not higher than 6%. It can be seen that the "other single components" are the most stringent of the requirements of the quality standard.
The "other single components" include both the avilamycin minor component and the associated impurities. The related substance detection method published by the bulletin is a high performance liquid chromatography-ultraviolet detection method, and the impurities are required to be detected by ultraviolet absorption at a selected wavelength (286nm), so that the impurities are at risk of being missed to be detected to a certain extent.
An Evaporative Light Scattering Detector (ELSD) is a universal type detector that eliminates the difficulties commonly found in conventional HPLC detection methods, unlike uv and fluorescence detectors, where the response is independent of the optical properties of the sample, is not affected by its functional groups, and any sample that is less volatile than the mobile phase can be detected, including materials that do not have uv absorption. The ELSD response value is proportional to the mass of the sample, and the detection of all samples has almost the same response factor, so that the ELSD response value can be used for determining the purity of the sample or detecting unknown substances. Differential detector (RI) is also a general-purpose detector, but has low sensitivity and is incompatible with gradient elution. Mass spectrometry is another general type of detector, but its application is limited by the expense and operational complexity.
Disclosure of Invention
In order to solve the above problems, the present invention provides a general detection method, which can detect impurities contained in a sample as much as possible, and effectively monitor the content of each component to strictly control the product quality. Therefore, the unique detection capability of ELSD is combined with the gradient elution separation capability of HPLC, and the separation degree is further optimized and improved on the basis of the original gradient condition, so that the method is used for detecting related substances of the avilamycin premix.
The technical scheme adopted for realizing the aim of the invention is that the method for detecting the avilamycin premix by HPLC-ELSD comprises the following steps:
a. preparing an avilamycin reference substance solution and a sample solution;
b. drawing a standard curve: respectively injecting 3-5 parts of reference substance solutions with different concentrations into HPLC-ELSD for determination, wherein the elution mode is gradient elution, and recording reference substance chromatogram and peak area; performing linear regression by using the logarithm of the peak area of the component A and the logarithm of the concentration value of the corresponding component A;
c. sample determination and result calculation: and (c) injecting the sample solution into HPLC-ELSD for determination, recording a chromatogram and a peak area, and respectively calculating the content percentages of A, B components and impurities in the chromatogram of the sample solution according to the linear regression equation obtained in the step b.
Further, the chromatographic conditions for HPLC-ELSD measurement were as follows:
a chromatographic column: c185.0 μm4.6 x 250 mm;
column temperature: 30-40 ℃;
sample introduction amount: 10-50 μ L;
flow rate: 0.9-1.1 mL/min;
temperature of the drift tube: 115 ℃ and 130 ℃;
gain factor: 1;
gas flow rate: 2.0-2.8L/min;
operating time: 40-50 min;
mobile phase A: methanol/ammonium acetate solution 50/50;
mobile phase B: methanol/ammonium acetate solution 80/20;
further, the gradient elution procedure is:
| time min | A％ | B％ |
| 0 | 70 | 30 |
| 3 | 70 | 30 |
| 37 | 35 | 65 |
| 40 | 70 | 30 |
| 45 | 70 | 30 |
Furthermore, the avilamycin reference solution takes acetonitrile/diammonium hydrogen phosphate buffer (1/1) as a solvent, and the concentration is 0.16-1.6 mg/mL.
Further, the preparation method of the avilamycin sample solution comprises the following steps: taking avilamycin and adding methanol to prepare a solution containing 0.8-1.3mg of avilamycin in each 1mL, fully stirring or performing ultrasonic treatment for 40-90min, shaking up, filtering by using filter paper, absorbing 5mL of filtrate and placing the filtrate in a culture dish, drying at room temperature in a fume hood, quantitatively adding acetonitrile/diammonium hydrogen phosphate buffer (1/1), stirring by using a glass rod to dissolve uniformly, and filtering by using a microporous filter membrane to obtain the avilamycin sample solution.
Further, the concentration of the ammonium acetate solution used in the mobile phase A and the mobile phase B was 0.04 mol/L.
Further, the processing method of the mobile phase A and the mobile phase B comprises the following steps: vacuum filtering methanol and ammonium acetate solution with organic and water system microporous filter membrane respectively, mixing at 50/50 and 80/20 ratio respectively to obtain mobile phase A and mobile phase B, and ultrasonic degassing for 15-30 min.
Further, the avilamycin sample solution is a premix sample solution.
Further, the pore size of the microporous filter membrane is 0.45 μm.
The invention has the beneficial effects that: the method is improved on the basis of the bulletin method, the leaching solvent is changed from acetone to methanol, and the toxicity and the volatility are reduced; the sample processing steps are simplified, and the efficiency is higher; the separation degree between peaks is improved after the gradient elution condition is optimized, the calculation of each component is more accurate, the pH value of a mobile phase does not need to be adjusted, the time is saved, and the possibility of misoperation is reduced; the detector is changed from an ultraviolet detector to a universal evaporative light scattering detector, the detection range is wider, and the monitoring on impurities is more comprehensive and stricter.
The method adopted by the invention has high accuracy, good repeatability and more comprehensive impurity monitoring, can be used as an internal control method of a production enterprise, enables the product quality to be higher than the quality standard, and has more advantages in the market.
Drawings
FIG. 1 is a chromatogram of a control solution of example 1 (mass concentration of 1.6 mg/mL);
FIG. 2 is a dichroism spectrum (mass concentration of 0.53mg/mL) of a control solution of example 1;
FIG. 3 is a trichromatography of a control solution of example 1 (mass concentration of 0.16 mg/mL);
FIG. 4 is a chromatogram of the sample solution of example 1 (calculated concentration of 0.98 mg/mL);
FIG. 5 is a chromatogram of a control solution of example 2 (mass concentration of 1.4 mg/mL);
FIG. 6 is a chromatogram of the sample solution of example 2 (calculated concentration of 0.53 mg/mL);
FIG. 7 is a chromatogram of a control solution of example 3 (mass concentration of 1.0mg/mL)
FIG. 8 is a chromatogram of the sample solution of example 3 (calculated concentration of 0.55 mg/mL);
FIG. 9 is a linear regression plot of an avilamycin control.
Detailed Description
The technical solution of the present invention is illustrated below by specific examples, but the scope of the present invention is not limited thereto:
1. instrument for measuring the position of a moving object
Liquid chromatograph: waters e2695
A detector: alltech ELSD 6000 evaporative light scattering detector
A chromatographic column: hubble C185 μm4.6 x 250mm
2. Gradient elution procedure:
| time min | A％ | B％ |
| 0 | 70 | 30 |
| 3 | 70 | 30 |
| 37 | 35 | 65 |
| 40 | 70 | 30 |
| 45 | 70 | 30 |
Specific example 1:
chromatographic conditions are as follows:
a chromatographic column: c185.0 μm4.6 x 250 mm;
column temperature: at 32 ℃;
sample introduction amount: 20 mu L of the solution;
flow rate: 1.1 mL/min;
temperature of the drift tube: 120 ℃;
gain factor: 1;
gas flow rate: 2.4L/min;
detection time: 45 min;
mobile phase A: methanol/ammonium acetate 50/50;
mobile phase B: methanol/ammonium acetate 80/20;
a. preparation of avilamycin control solutions: accurately weighing 40mg of avilamycin reference substance, dissolving in 25mL of acetonitrile/diammonium phosphate buffer (1/1) mixed solvent, and shaking up to obtain a reference substance solution I; respectively diluting the reference substance solution by 3 times and 10 times with mixed solvent of acetonitrile/diammonium hydrogen phosphate buffer solution (1/1) to obtain reference substance solutions II and III;
b. preparation of avilamycin sample solution: taking a proper amount of an avilamycin sample, adding methanol to prepare a solution containing 1.0mg of avilamycin in every 1mL, fully stirring for 40min, shaking up, filtering by using filter paper, absorbing 5mL of filtrate, placing the filtrate in a culture dish, airing at room temperature in a fume hood, adding 5mL of acetonitrile/diammonium hydrogen phosphate buffer (1/1), stirring by using a glass rod to dissolve uniformly, and filtering by using a microporous filter membrane to obtain a test solution;
c. preparing a standard curve: opening a liquid chromatograph, balancing a base line for 20min after the instrument parameters meet the requirements, sequentially injecting reference substance solutions III, II and I, wherein each solution is injected in parallel for 2 times, and the separation degree R of a target peak and front and rear impurity peaks is more than 1.5; chromatogram and peak area were recorded. Performing linear regression by using the logarithm of the peak area of the component A and the logarithm of the concentration value of the corresponding component A;
d. sample determination and result calculation: and (c) injecting the sample solution into HPLC-ELSD for determination, recording a chromatogram and a peak area, and respectively calculating the content percentages of A, B components and various impurities in the chromatogram of the sample solution according to the linear regression equation obtained in the step c.
As can be seen from FIGS. 1 to 4, a chromatogram (mass concentration of 1.6mg/mL) of the control solution of example 1; retention time A34.411 min, retention time B24.307 min. Example 1 control solution dichroism spectrum (mass concentration of 0.53 mg/mL); retention time A34.435 min, retention time B24.262 min. Example 1 trichromatography of control solution (0.16 mg/mL); retention time A34.451 min, retention time B24.295 min. Example 1 sample solution chromatogram (calculated concentration of 0.98 mg/mL); retention time A34.427 min, retention time B24.294 min.
Specific example 2:
chromatographic conditions are as follows:
a chromatographic column: c185.0 μm4.6 x 250 mm;
column temperature: 35 ℃;
sample introduction amount: 30 mu L of the solution;
flow rate: 1.0 mL/min;
temperature of the drift tube: 118 ℃;
gain factor: 1;
gas flow rate: 2.2L/min;
detection time: 45 min;
mobile phase A: methanol/ammonium acetate 50/50;
mobile phase B: methanol/ammonium acetate 80/20;
a. preparation of avilamycin control solutions: accurately weighing 70mg of avilamycin reference substance, dissolving in 50mL of acetonitrile/diammonium hydrogen phosphate buffer (1/1) mixed solvent, and shaking up to obtain a reference substance solution I; respectively diluting the reference substance solution by 5 times and 8 times with mixed solvent of acetonitrile/diammonium hydrogen phosphate buffer solution (1/1) to obtain reference substance solutions II and III;
b. preparation of avilamycin sample solution: taking a proper amount of an avilamycin sample, adding methanol to prepare a solution containing 1.1mg of avilamycin in every 1mL, carrying out ultrasonic treatment for 50min, shaking up, filtering by using filter paper, sucking 5mL of filtrate, placing the filtrate in a culture dish, airing the filtrate at room temperature in a fume hood, adding 10mL of acetonitrile/diammonium phosphate buffer (1/1), stirring by using a glass rod to dissolve the solution uniformly, and filtering the solution by using a microporous filter membrane to obtain a test solution;
c. preparing a standard curve: opening a liquid chromatograph, balancing a base line for 30min after the instrument parameters meet the requirements, sequentially injecting reference substance solutions III, II and I, wherein each solution is injected in parallel for 3 times, and the separation degree R of a target peak and front and rear impurity peaks is more than 1.5; chromatogram and peak area were recorded. Performing linear regression by using the logarithm of the peak area of the component A and the logarithm of the concentration value of the corresponding component A;
d. sample determination and result calculation: and (c) injecting the sample solution into HPLC-ELSD for determination, recording a chromatogram and a peak area, and respectively calculating the content percentages of A, B components and various impurities in the chromatogram of the sample solution according to the linear regression equation obtained in the step c.
As shown in FIGS. 5 to 6, a chromatogram (mass concentration of 1.4mg/mL) of the control solution of example 2; retention time A34.540 min, retention time B24.415 min. Example 2 sample solution chromatogram (calculated concentration of 0.53 mg/mL); retention time A34.416 min, retention time B24.249 min.
Specific example 3:
chromatographic conditions are as follows:
a chromatographic column: c185.0 μm4.6 x 250 mm;
column temperature: 38 ℃;
sample introduction amount: 25 mu L of the solution;
flow rate: 0.9 mL/min;
temperature of the drift tube: 125 ℃;
gain factor: 1;
gas flow rate: 2.6L/min;
detection time: 50 min;
mobile phase A: methanol/ammonium acetate 50/50;
mobile phase B: methanol/ammonium acetate 80/20;
a. preparation of avilamycin control solutions: accurately weighing 50mg of avilamycin reference substance, dissolving in 50mL of acetonitrile/diammonium hydrogen phosphate buffer (1/1) mixed solvent, and shaking up to obtain a reference substance solution I; respectively diluting the reference substance solution by 2 times and 4 times with mixed solvent of acetonitrile/diammonium hydrogen phosphate buffer solution (1/1) to obtain reference substance solutions II and III;
b. preparation of avilamycin sample solution: taking a proper amount of an avilamycin sample, adding methanol to prepare a solution containing 1.2mg of avilamycin in every 1mL, carrying out ultrasonic treatment for 80min, shaking up, filtering by using filter paper, sucking 5mL of filtrate, placing the filtrate in a culture dish, airing the filtrate at room temperature in a fume hood, adding 10mL of acetonitrile/diammonium phosphate buffer (1/1), stirring by using a glass rod to dissolve the solution uniformly, and filtering the solution by using a microporous filter membrane to obtain a test solution;
c. preparing a standard curve: opening a liquid chromatograph, balancing a base line for 40min after the instrument parameters meet the requirements, sequentially injecting reference substance solutions III, II and I, wherein each solution is injected in parallel for 3 times, and the separation degree R of a target peak and front and rear impurity peaks is more than 2.0; chromatogram and peak area were recorded. Performing linear regression by using the logarithm of the peak area of the component A and the logarithm of the concentration value of the corresponding component A;
d. sample determination and result calculation: and (c) injecting the sample solution into HPLC-ELSD for determination, recording a chromatogram and a peak area, and respectively calculating the content percentages of A, B components and various impurities in the chromatogram of the sample solution according to the linear regression equation obtained in the step c.
As shown in fig. 7-8, example 3 has a chromatogram of a control solution (mass concentration of 1.0 mg/mL); retention time A35.190 min, retention time B24.860 min. Example 3 sample solution chromatogram (calculated concentration of 0.55 mg/mL); retention time A36.222 min, retention time B25.843 min.
Claims (4)
1. A method for detecting an avilamycin premix by HPLC-ELSD is characterized by comprising the following steps: the method comprises the following steps:
a. preparing an avilamycin reference substance solution and a sample solution;
b. drawing a standard curve: respectively injecting 3-5 parts of reference substance solutions with different concentrations into HPLC-ELSD for determination, wherein the elution mode is gradient elution, and recording reference substance chromatogram and peak area; performing linear regression by using the logarithm of the peak area of the component A and the logarithm of the concentration value of the corresponding component A;
c. sample determination and result calculation: injecting the sample solution into HPLC-ELSD for determination, recording the chromatogram and the peak area, and respectively calculating the content percentages of A, B components and impurities in the chromatogram of the sample solution according to the linear regression equation obtained in the step b;
the chromatographic conditions for HPLC-ELSD are as follows:
a chromatographic column: c185.0 μm4.6 x 250 mm;
column temperature: 30-40 ℃;
sample introduction amount: 10-50 μ L;
flow rate: 0.9-1.1 mL/min;
temperature of the drift tube: 115 ℃ and 130 ℃;
gain factor: 1;
gas flow rate: 2.0-2.8L/min;
operating time: 40-50 min;
mobile phase A: methanol/ammonium acetate solution = 50/50;
mobile phase B: methanol/ammonium acetate solution = 80/20;
the avilamycin reference solution takes acetonitrile/diammonium hydrogen phosphate buffer solution 1/1 as a solvent, and the concentration is 0.16-1.6 mg/mL;
the preparation method of the avilamycin sample solution comprises the following steps: preparing avilamycin into a solution containing 0.8-1.3mg of avilamycin in each 1mL of the avilamycin, fully stirring or ultrasonically treating for 40-90min, shaking up, filtering by using filter paper, absorbing 5mL of filtrate, placing the filtrate into a culture dish, airing at room temperature in a fume hood, quantitatively adding acetonitrile/diammonium hydrogen phosphate buffer 1/1, stirring by using a glass rod to uniformly dissolve, and filtering by using a microporous filter membrane to obtain an avilamycin sample solution;
the processing method of the mobile phase A and the mobile phase B comprises the following steps: vacuum filtering methanol and ammonium acetate solution with organic system and water system microporous filter membrane respectively, mixing at 50/50 and 80/20 ratio respectively to obtain mobile phase A and mobile phase B, and ultrasonic degassing for 15-30 min;
the gradient elution procedure was:
。
2. The method for detecting avilamycin premix by HPLC-ELSD as claimed in claim 1, wherein: the concentration of the ammonium acetate solution used in the mobile phase A and the mobile phase B is 0.04 mol/L.
3. The method for detecting avilamycin premix by HPLC-ELSD as claimed in claim 1, wherein: the avilamycin sample solution is a premix sample solution.
4. The method for detecting avilamycin premix by HPLC-ELSD as claimed in claim 1, wherein: the aperture of the microporous filter membrane is 0.45 mu m.
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